Methods for treatment of type I diabetes

ABSTRACT

Substituted condensation products of -benzyl-3-indenylacetamides with heterocyclic aldehydes and other such inhibitors are useful for the treatment of type I diabetes.

TECHNICAL FIELD

This invention relates to the treatment of type I diabetes.

BACKGROUND OF THE INVENTION

Type 1 or Insulin Dependent Diabetes Mellitus (IDDM) is estimated toaffect 675,000 people in the United States. This represents 0.3% of thetotal U.S. population, with an estimated 30,000 new cases diagnosed eachyear. Complications of diabetes impair the longevity and quality oflife, and include atherosclerotic heart disease, gangrene and stroke, aswell as diabetic retinopathy, neuropathy and nephropathy. Within 15years after the onset of diabetes, retinopathy may be observed in 97% ofType 1 diabetics. Today diabetic retinopathy remains the leading causeof blindness in the U.S. and patients with diabetes are 25 times morelikely to develop blindness than the general population.

Symptoms of diabetic neuropathy have been observed in 54% of Type 1patients studied. Patients present with symptoms ranging from peripheralsensory-deficits (pins and needles/carpal tunnel syndrome) to autonomicneuropathy resulting in bladder and bowel dysfunction. Type 1 diabetesis also responsible for a large proportion of the patients on renaldialysis, the result of diabetes-induced end stage renal disease. Morethan 40% of Type 1 patients who have had diabetes for more than 20 yearshave diabetic nephropathy. The prevalence of myocardial infarction,angina and stroke is 2-3 times greater than in non-diabetics, and theType 1 diabetic's life span is shortened by about 15 years. Theestimated total cost of Type 1 diabetes in the United States (medicalexpenses, lost wages etc.) is greater than $20 billion per year.

Type I diabetes actually begins before the clinical manifestations ofthe disease. It starts with the progressive destruction of beta cells inthe pancreas. These cells normally produce insulin. The reduction ofinsulin response to glucose can be measured during this period, however.Ultimately there is massive (>90%) destruction of beta cells in theislets of Langer hans. During the early stages of the disease andbeyond, type I diabetes is characterized by the infiltration ofpancreatic islets by macrophages and lymphocytes (helper and killer).The macrophage infiltration is believed to prompt the infiltration ofsmall lymphocytes. While clinicians understand the potential for a drugthat can address macrophage involvement early in the disease, no safetherapies have yet been found. Current treatment involves daily frequentinjections of insulin. However, this can lead to side effects such ashypoglycemic shock.

Thus, there is an urgent, unmet need for safe and effective drugtherapies for type I diabetes, particularly a therapy that can intervenein the early stages of the disease to treat macrophage infiltration andprevent subsequent beta cell damage by macrophages.

SUMMARY OF THE INVENTION

This invention represents a novel therapy for treating patients (e.g.,humans or companion animals) with type I diabetes without thesubstantial side effects of prior pharmaceutical approaches.Specifically, this invention involves the administration of an inhibitorof phosphodiesterase 2 (“PDE2”) to a mammal in need of treatment fortype I diabetes. Preferably, that inhibitor also inhibitsphosphodiesterase 5 (“PDE5”). In narrower aspects of this invention,this invention involves the administration of compounds of Formula Ibelow to a mammal in need of treatment for type I diabetes.

As explained below, compounds that inhibit PDE2 can cause activatedmacrophages to undergo apoptosis.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a graph that compares the PDE2 and PDE5 MRNA levels in controland activated macrophages.

FIG. 2 is a fluorescent microscope photomicrograph of controlmacrophages stained via indirect immunofluorescence to show basal levelof PDE5 protein in the cells.

FIG. 3 is a fluorescent microscope photomicrograph of activatedmacrophages stained via indirect immunofluorescence to show increasedlevel of PDE5 protein in the cells.

FIG. 4 is a fluorescent microscope photomicrograph of controlmacrophages stained via indirect immunofluorescence to show basal levelof PDE2 protein in the cells.

FIG. 5 is a fluorescent microscope photomicrograph of activatedmacrophages stained via indirect immunofluorescence to show increasedlevel of PDE2 protein in the cells.

FIG. 6 is a graph that illustrates cGMP and cAMP hydrolysis levels inactivated and control macrophages.

FIG. 7 is a graph that illustrates cGMP hydrolysis levels in proteinlysates from activated and control macrophages.

FIG. 8 is a digital image obtained with a fluorescent microscope ofactivated macrophages treated with a PDE2 inhibitor wherein themacrophages undergo apoptosis as reflected by the presence of activecaspase 3 (red signal).

FIG. 9 is a digital image obtained with a fluorescent microscope ofcontrol (vehicle only) macrophages revealing only low, background levelsof apoptosis as reflected by the reduced presence of active caspase 3(red signal).

FIG. 10 is a digital image obtained with a fluorescent microscope ofactivated macrophages treated with a PDE4-specific inhibitor wherein themacrophages do not undergo substantial apoptosis as reflected by thesubstantial absence of active caspase 3 (red signal).

FIG. 11 is a digital image obtained with a fluorescent microscope ofactivated macrophages treated with a PDE5-specific inhibitor wherein themacrophages do not undergo substantial apoptosis as reflected by thesubstantial absence of active caspase 3 (red signal).

FIG. 12 is a graph illustrating decreased TNFα levels in activatedmacrophages with exposure to a PDE2 inhibitor.

FIG. 13a is a photomicrograph of a regional islet zone in a BBDP/Wor rat(prone diabetic) pancreatic section where the positive brown stainedtissue denotes activated macrophage homing toward the islet zone in adilated capillary (arrowhead).

FIG. 13b is a photomicrograph of a regional islet zone in a BBDRJWor rat(diabetic resistant) pancreatic section (arrow). This section is from anaged-matched control for FIG. 13a.

FIG. 14a is a photomicrograph of a regional islet zone in a BBDP/Wor rat(acute diabetic) pancreatic section where the positive brown stainedtissue denotes activated macrophage in the islet region (arrowhead).

FIG. 14b is a photomicrograph of a regional islet zone in a BBDR/Wor rat(diabetic resistant) pancreatic section (arrow). This section is from anaged-matched control for FIG. 14a.

FIG. 15a is a photomicrograph of a regional islet zone in a BBDP/Wor rat(chronic diabetic) pancreatic section where the positive brown stainedtissue denotes activated macrophage in the islet region (arrowhead).

FIG. 15b is a photomicrograph of a regional islet zone in a BBDR/Wor rat(diabetic resistant) pancreatic section (arrow). This section is from anaged-matched control for FIG. 15a.

FIG. 16 is a bar graph that denotes the total number of positive stainedtissue-activated rat macrophages in eight randomly counted isletcellular regions. Note the significant greater numbers of macrophagesare present in the acute BBDP/W or rat pancreas.

FIGS. 17a and 17 b represent pancreatic tissue specimens from two humanpatients where the specimens are stained for PDE2 protein.

FIGS. 18a and 18 b represent pancreatic tissue specimens from two humanpatients where the specimens are stained for PDE5 protein.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention includes the administration ofan inhibitor of PDE2 to a mammal in need of treatment for type Idiabetes. In addition, this invention includes the use of compounds ofFormula I below (as well as their pharmaceutically acceptable salts) fortreating a mammal with type I diabetes:

wherein R¹ is independently selected in each instance from the groupconsisting of hydrogen, halogen, lower alkyl, lower alkoxy, amino, loweralkylamino, di-lower alkylamino, lower alkylmercapto, lower alkylsulfonyl, cyano, carboxamide, carboxylic acid, mercapto, sulfonic acid,xanthate and hydroxy; R₂ is selected from the group consisting ofhydrogen and lower alkyl;

R₃ is selected from the group consisting of hydrogen, halogen, amino,hydroxy, lower alkyl amino, and di-loweralkylamino;

R₄ is hydrogen, or R₃ and R₄ together are oxygen;

R₅ and R₆ are independently selected from the group consisting ofhydrogen, lower alkyl, hydroxy-substituted lower alkyl, amino loweralkyl, lower alkylamino-lower alkyl, lower alkyl amino di-lower alkyl,lower alkyl nitrile, —CO₂H, —C(O)NH₂, and a C₂ to C₆ amino acid;

R₇ is independently selected in each instance from the group consistingof hydrogen, amino lower alkyl, lower alkoxy, lower alkyl, hydroxy,amino, lower alkyl amino, di-lower alkyl amino, halogen, —CO₂H, —SO₃H,—SO₂NH₂, and —SO₂(lower alkyl);

m and n are integers from 0 to 3 independently selected from oneanother;

Y is selected from the group consisting of quinolinyl, isoquinolinyl,pyridinyl, pyrinidiyl, pyrazinyl, imidazolyl, indolyl, benzimidazolyl,triazinyl, tetrazolyl, thiophenyl, furanyl, thiazolyl, pyrazolyl, orpyrrolyl, or subsituted variants thereof wherein the substituents areone or two selected from the group consisting of halogen, lower alkyl,lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy,—SO₂(lower alkyl) and —SO₂NH₂.

Preferred compounds of this invention for use with the methods describedherein include those of Formula I where:

R₁ is selected from the group consisting of halogen, lower alkoxy,amino, hydroxy, lower alkylamino and di-loweralkylamino, preferablyhalogen, lower alkoxy, amino and hydroxy;

R₂ is lower alkyl;

R₃ is selected from the group consisting of hydrogen, halogen, hydroxy,amino, lower alkylamino and di-loweralkylamino, preferably, hydrogen,hydroxy and lower alkylamino;

R₅ and R₆ are independently selected from the group consisting ofhydrogen, hydroxy-substituted lower alkyl, amino lower alkyl, loweralkylamino-lower alkyl, lower alkyl amino di-lower alkyl, —CO₂H,—C(O)NH₂; preferably hydrogen, hydroxy-substituted lower alkyl, loweralkyl amino di-lower alkyl, —CO₂H, and —C(O)NH₂;

R₇ is independently selected in each instance from the group consistingof hydrogen, lower alkoxy, hydroxy, amino, lower alkyl amino, di-loweralkyl amino, halogen, —CO₂H, —SO₃H, —SO₂NH₂, and —SO₂(lower alkyl);preferably hydrogen, lower alkoxy, hydroxy, amino, amino lower alkyl,halogen, —CO₂H, —SO₃H, —SO₂NH₂, and —SO₂(lower alkyl);

Preferably, at least one of the R₇ substituents is para- orortho-located; most preferably ortho-located;

Y is selected from the group consisting of quinolinyl, isoquinolinyl,pyridinyl, pyrimidinyl and pyrazinyl or said substituted variantsthereof.

Preferably, the substituents on Y are one or two selected from the groupconsisting of lower alkoxy, amino, lower alkylamino, di-loweralkylamino, hydroxy, —SO₂(lower alkyl) and —SO₂NH₂; most preferablylower alkoxy, di-lower alkylamino, hydroxy, —SO₂(lower alkyl) and—SO₂NH₂.

The present invention also is a method of treating a mammal with type Idiabetes by administering to a patient a pharmacologically effectiveamount of a pharmaceutical composition that includes a compound ofFormula I, wherein R₁ through R₇ and Y are as defined above. Preferably,this composition is administered without therapeutic amounts of anNSAID.

Compounds of this invention are inhibitors of phosphodiesterases PDE2.For convenience, the PDE inhibitory activity of such compounds can betested as taught in U.S. patent application Ser. No. 09/046,739 filedMar. 24, 1998 to Pamukcu et al., which is incorporated herein byreference. Thus, compounds employed in this invention are usefulinhibitors of PDE2 and preferably also PDE5. Most preferably, suchcompounds have an IC50 for PDE2 of no more than 25 μM.

Additional compounds besides those of Formula I can be identified forinhibitory effect on the activity of PDE2 and/or PDE5. Alternatively,cyclic nucleotide levels in whole cells are measured by radioimmunoassay(“RIX”) and compared to untreated and drug-treated tissue samples and/orisolated enzymes.

Phosphodiesterase activity can be determined using methods known in theart, such as a method using radioactive ³H cyclic GMP (cGMP)(cyclic3′,5′-guanosine monophosphate) as the substrate for the PDE enzyme.(Thompson, W. J., Teraski, W. L., Epstein, P. M., Strada, S. J.,Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which isincorporated herein by reference). In brief, a solution of definedsubstrate ³H-cGMP specific activity (0.2 μM; 100,000 cpm; containing 40mM Tris-HCl (pH 8.0), 5 mM MgCl₂ and 1 mg/mL BSA) is mixed with the drugto be tested in a total volume of 400 μl. The mixture is incubated at30° C. for 10 minutes with isolated PDE2 and/or PDE5. Reactions areterminated, for example, by boiling the reaction mixture for 75 seconds.After cooling on ice, 100 μl of 0.5 mg/mL snake venom (O. Hannah venomavailable from Sigma) is added and incubated for 10 minutes at 30° C.This reaction is then terminated by the addition of an alcohol, e.g. 1mL of 100% methanol. Assay samples are applied to 1 mL Dowex 1-X8column; and washed with 1 mL of 100% methanol. The amount ofradioactivity in the breakthrough and the wash from the column iscombined and measured with a scintillation counter. The degree ofphosphodiesterase inhibition is determined by calculating the amount ofradioactivity in drug-treated reactions and comparing against a controlsample (a reaction mixture lacking the tested compound but with drugsolvent).

Alternatively, the ability of desirable compounds to inhibit thephosphodiesterases of this invention is reflected by an increase in cGMPin type I diabetes (pancreatic) tissue samples exposed to a compoundbeing evaluated. The amount of PDE activity can be determined byassaying for the amount of cyclic GMP in the extract of treated cellsusing RIA. When PDE activity is evaluated in this fashion, a combinedcGMP hydrolytic activity is assayed. The test compound is then incubatedwith the tissue at a concentration of compound between about 200 μM toabout 200 μM. About 24 to 48 hours thereafter, the culture media isremoved from the tissue, and the cells are solubilized. The reaction isstopped by using 0.2N HCl/50% MeOH. A sample is removed for proteinassay. Cyclic GMP is purified from the acid/alcohol extracts of cellsusing anion-exchange chromatography, such as a Dowex column. The cGMP isdried, acetylated according to published procedures, such as usingacetic anhydride in triethylamine, (Steiner, A. L., Parker, C. W.,Kipnis, D. M., J. Biol. Chem., 247(4):1106-13, 1971, which isincorporated herein by reference). The acetylated cGMP is quantitatedusing radioimmunoassay procedures (Harper, J., Brooker, G., Advances inNucleotide Research, 10: 1-33, 1979, which is incorporated herein byreference). Iodinated ligands (tyrosine methyl ester) of derivatizedcyclic GMP are incubated with standards or unknowns in the presence ofantisera and appropriate buffers. Antiserum may be produced using cyclicnucleotide-haptene directed techniques. The antiserum is from sheepinjected with succinyl-cGMP-albumin conjugates and diluted 1/20,000.Dose-interpolation and error analysis from standard curves are appliedas described previously (Seibert, A. F., Thompson, W. J., Taylor, A.,Wilbourn, W. H., Barnard, J. and Haynes, J., J. Applied Physiol.,72:389-395, 1992, which is incorporated herein by reference).

In addition, the tissue may be acidified, frozen (−70° C.) and alsoanalyzed for cGMP and cAMP.

More specifically as to tissue testing, the PDE inhibitory activityeffect of a compound can also be determined from tissue biopsiesobtained from humans or tissues from animals exposed to the testcompound. A sample of tissue is homogenized in 500 μl of 6%trichloroacetic acid (“TCA”). A known amount of the homogenate isremoved for protein analysis. The remaining homogenate is allowed to siton ice for 20 minutes to allow for the protein to precipitate. Next, thehomogenate is centrifuged for 30 minutes at 15,000 g at 4° C. Thesupernatant is recovered, and the pellet recovered. The supernatant iswashed four times with five volumes of water saturated diethyl ether.The upper ether layer is discarded between each wash. The aqueous etherextract is dried in a speed vac. Once dried, the sample can be frozenfor future use, or used immediately. The dried extract is dissolved in500 μl of assay buffer. The amount of PDE inhibition is determined byassaying for the amount of cyclic nucleotides using RIA procedures asdescribed above.

In addition to compounds disclosed herein, other compounds that inhibitboth PDE2 and PDE5 include compounds disclosed in U.S. Pat. Nos.5,401,774 (e.g., exisulind), 6,063,818, 5,998,477, and 5,965,619. Thesepatents are incorporated herein by reference.

When referring to an “a physiologically effective amount of an inhibitorof PDE2 and PDE5” we mean not only a single compound that inhibits thoseenzymes but a combination of several compounds, each of which caninhibit one or both of those enzymes. Single compounds that inhibit bothenzymes are preferred.

When referring to an “inhibitor [that] does not substantially inhibitCOX I or COX II,” we mean that in the ordinary sense of the term. By wayof example only, if the inhibitor has an IC₅₀ for either PDE2 or PDE5that is at least half of the IC₅₀ of COXI and/or COXII, a drug achievingthe PDE IC₅₀ in the blood could be said not to substantially inhibit theCOX enzymes. Preferably, the IC₅₀ for the COX enzymes is in the order of10 fold or more higher than the IC₅₀ for PDE2/PDE5. Preferably, the IC₅₀for the COX enzymes is greater than about 40 μM.

As used herein, the term “halo” or “halogen” refers to chloro, bromo,fluoro and iodo groups, and the term “alkyl” refers to straight,branched or cyclic alkyl groups and to substituted aryl alkyl groups.The term “lower alkyl” refers to C₁ to C₈ alkyl groups.

The term “hydroxy-substituted lower alkyl” refers to lower alkyl groupsthat are substituted with at least one hydroxy group, preferably no morethan three hydroxy groups.

The term “—SO₂(lower alkyl)” refers to a sulfonyl group that issubstituted with a lower alkyl group.

The term “lower alkoxy” refers to alkoxy groups having from 1 to 8carbons, including straight, branched or cyclic arrangements.

The term “lower alkylmercapto” refers to a sulfide group that issubstituted with a lower alkyl group; and the term “lower alkylsulfonyl” refers to a sulfone group that is substituted with a loweralkyl group.

The term “pharmaceutically acceptable salt” refers to non-toxic acidaddition salts and alkaline earth metal salts of the compounds ofFormula I. The salts can be prepared in situ during the final isolationand purification of such compounds, or separately by reacting the freebase or acid functions with a suitable organic acid or base, forexample. Representative acid addition salts include the hydrochloride,hydrobromide, sulfate, bisulfate, acetate, valerate, oleate, palmatate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,mesylate, citrate, maleate, fumarate, succinate, tartrate,glucoheptonate, lactobionate, lauryl sulfate salts and the like.Representative alkali and alkaline earth metal salts include the sodium,calcium, potassium and magnesium salts.

It will be appreciated that certain compounds of Formula I can possessan asymmetric carbon atom and are thus capable of existing asenantiomers. Unless otherwise specified, this invention includes suchenantiomers, including any racemates. The separate enaniomers may besynthesized from chiral starting materials, or the racemates can beresolved by conventional procedures that are well known in the art ofchemistry such as chiral chromatography, fractional cyrstallization ofdiastereomeric salts and the like.

Compounds of Formula I also can exist as geometrical isomers (Z and E);the Z isomer is preferred.

Compounds of this invention may be formulated into pharmaceuticalcompositions together with pharmaceutically acceptable carriers for oraladministration in solid or liquid form, or for rectal or topicaladministration, although carriers for oral administration are mostpreferred.

Pharmaceutically acceptable carriers for oral administration includecapsules, tablets, pills, powders, troches and granules. In such soliddosage forms, the carrier can comprise at least one inert diluent suchas sucrose, lactose or starch. Such carriers can also comprise, as isnormal practice, additional substances other than diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, troches and pills, the carriers may also comprise bufferingagents. Carriers such as tablets, pills and granules can be preparedwith enteric coatings on the surfaces of the tablets, pills or granules.Alternatively, the enterically coated compound can be pressed into atablet, pill, or granule, and the tablet, pill or granules foradministration to the patient. Preferred enteric coatings include thosethat dissolve or disintegrate at colonic pH such as shellac or EudragetS.

Pharmaceutically acceptable carriers include liquid dosage forms fororal administration, e.g., pharmaceutically acceptable emulsions,solutions, suspensions, syrups and elixirs containing inert diluentscommonly used in the art, such as water. Besides such inert diluents,compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring andperfuming agents.

Pharmaceutically acceptable carriers for topical administration includeDMSO, alcohol or propylene glycol and the like that can be employed withpatches or other liquid-retaining material to hold the medicament inplace on the skin so that the medicament will not dry out.

Pharmaceutically acceptable carriers for rectal administration arepreferably suppositories that may contain, in addition to the compoundsof this invention excipients such as cocoa butter or a suppository wax,or gel.

The pharmaceutically acceptable carrier and compounds of this inventionare formulated into unit dosage forms for administration to a patient.The dosage levels of active ingredient (i.e., compounds of thisinvention) in the unit dosage may be varied so as to obtain an amount ofactive ingredient effective to achieve lesion-eliminating activity inaccordance with the desired method of administration (i.e., oral orrectal). The selected dosage level therefore depends upon the nature ofthe active compound administered, the route of administration, thedesired duration of treatment, and other factors. If desired, the unitdosage may be such that the daily requirement for active compound is inone dose, or divided among multiple doses for administration, e.g., twoto four times per day.

The compounds of this invention can be formulated with pharmaceuticallyacceptable carriers into unit dosage forms in a conventional manner sothat the patient in need of therapy for type I diabetes can periodically(e.g., once or more per day) take a compound according to the methods ofthis invention. The exact initial dose of the compounds of thisinvention can be determined with reasonable experimentation. The initialdosage calculation would also take into consideration several factors,such as the formulation and mode of administration, e.g. oral orintravenous, of the particular compound. A total daily oral dosage ofabout 50 mg-2.0 gr of such compounds would achieve a desired systemiccirculatory concentration. As discussed below, an oral dose of about 800mg/day has been found appropriate in mammals.

As explained below, PDE2/5 inhibitors within this invention causeapoptosis in the infiltrating, activated macrophages that are associatedwith damage to pancreatic β-islet cells. Because the diabetic patientcontinually produces activated macrophages, PDE2/5 inhibitors of thisinvention should be administered chronically, i.e., for at least twoweeks at a time. hi this manner, as any activated macrophages arise frommonocytes, the drug in the patient's system can cause the macrophages toapoptose. In our hands, PDE2/5 inhibitors do cause monocytes toapoptose, given that animals exposed to such inhibitors chronically havenormal monocyte blood counts.

The pharmaceutical compositions of this invention are preferablypackaged in a container (e.g., a box or bottle, or both) with suitableprinted material (e.g., a package insert) containing indications anddirections for use in the treatment of type I diabetes, etc.

There are several general schemes for producing compounds of Formula Iuseful in this invention. One general scheme (which has severalsub-variations) involves the case where both R₃ and R₄ are bothhydrogen. This first scheme is described immediately below in Scheme I.The other general scheme (which also has several sub-variations)involves the case where at least one of R₃ and R₄ is a moiety other thanhydrogen but within the scope of Formula I above. This second scheme isdescribed below as “Scheme II.”

The general scheme for preparing compounds where both R₃ and R₄ are bothhydrogen is illustrated in Scheme I, which is described in part in U.S.Pat. No. 3,312,730, which is incorporated herein by reference. In SchemeI, R₁ is as defined in Formula I above. However, in Scheme I, thatsubstituent can also be a reactive moiety (e.g. a nitro group) thatlater can be reacted to make a large number of other substituted indenesfrom the nitro-substituted indenes.

In Scheme I, several sub-variations can be used. In one sub-variation, asubstituted benzaldehyde (a) may be condensed with a substituted aceticester in a Knoevenagel reaction (see reaction 2) or with an a-halogenopropionic ester in a Reformatsky Reaction (see reactions 1 and 3). Theresulting unsaturated ester (c) is hydrogenated and hydrolyzed to give asubstituted benzyl propionic acid (e) (see reactions 4 and 5).Alternatively, a substituted malonic ester in a typical malonic estersynthesis (see reactions 6 and 7) and hydrolysis decarboxylation of theresulting substituted ester (g) yields the benzyl propionic acid (e)directly. This latter method is especially preferable for nitro andalkylthio substituents on the benzene ring.

The next step is the ring closure of the β-aryl proponic acid (e) toform an indanone (h) which may be carried out by a Friedel-CraftsReaction using a Lewis acid catalyst (Cf. Organic Reactions, Vol. 2, p.130) or by heating with polyphosphoric acid (see reactions 8 and 9,respectively). The indanone (h) may be condensed with an a-halo ester inthe Reformatsky Reaction to introduce the aliphatic acid side chain byreplacing the carboxyl group (see reaction 10). Alternately, thisintroduction can be carried out by the use of a Wittig Reaction in whichthe reagent is a α-triphenylphosphinyl ester, a reagent that replacesthe carbonyl with a double bond to the carbon (see reaction 12). Thisproduct (1) is then immediately rearranged into the indene (j)(seereaction 13). If the Reformatsky Reaction route is used, theintermediate 3-hydroxy-3-aliphatic acid derivative i must be dehydratedto the indene (j) (see reaction 11).

The indenylacetic acid (k) in THF then is allowed to react with oxalylor thionyl chloride or similar reagent to produce the acid chloride (m)(see reaction 15), whereupon the solvent is evaporated. There are twomethods to carry out reaction 16, which is the addition of thebenzylamine side chain (n).

Method (I)

In the first method, the benzylamine (n) is added slowly at roomtemperature to a solution of 5-fluoro-2-methyl-3-indenylacetyl chloridein CH₂Cl₂. The reaction mixture is refluxed overnight, and extractedwith aqueous HCl (10%), water, and aqueous NaHCO₃ (5%). The organicphase is dried (Na₂SO₄) and is evaporated to give the amide compound(o).

Method (II)

In the second method, the indenylacetic acid (k) in DMA is allowed toreact with a carbodiimide (e.g.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) andbenzylamine at room temperature for two days. The reaction mixture isadded dropwise to stirred ice water. A yellow precipitate is filteredoff, is washed with water, and is dried in vacuo. Recrystallizationgives the amide compound (o).

Compounds of the type a′ (Scheme III), o (Scheme I), t (Scheme II), y(Scheme IIB) may all be used in the condensation reaction shown inScheme III.

Substituents

X=halogen, usually Cl or Br.

E=methyl, ethyl or benzyl, or lower acyl.

R₁, R₂, R₆, R₅, and R₇=as defined in Formula I.

Y, n and m=as defined in Formula I.

Reagents and general conditions for Scheme I (numbers refer to thenumbered reactions):

(1) Zn dust in anhydrous inert solvent such as benzene and ether.

(2) KHSO₄ or p-toluene sulfonic acid.

(3) NaOC₂H₅ in anhydrous ethanol at room temperature.

(4) H₂ palladium on charcoal, 40 p.s.i. room temperature.

(5) NaOH in aqueous alcohol at 20-100°.

(6) NaOC₂H₅ or any other strong base such as NaH or K-t-butoxide.

(7) Acid.

(8) Friedel-Crafts Reaction using a Lewis Acid catalyst Cf. OrganicReactions, Vol. II, p. 130.

(9) Heat with polyphosphoric acid.

(10) Reformatsky Reaction: Zn in inert solvent, heat.

(11) p-Toluene sulfonic acid and CaCl₂ or I₂ at 200°

(12) Wittig Reaction using (C₆H₅)₃ P═C—COOE 20-80° in ether or benzene

(13) (a) NBS/CCl₄/benzoyl peroxide

(b) PtO₂/H₂ (1 atm.)/acetic acid

(14) (a) NaOH

(b) HCl

(15) Oxalyl or thionyl chloride in CH₂Cl₂ or THF

(16) Method I: 2 equivalents of NH₂—C(R₅R₆)—Ph—(R₇)_(m)

Method II: carbodiimide in THF

(17) 1N NaOCH₃ in MeOH under reflux conditions

Indanones within the scope of compound (h) in Scheme I are known in theliterature and are thus readily available as intermediates for theremainder of the synthesis so that reactions 1-7 can be convenientlyavoided. Among such known indanones are:

5-methoxyindanone

6-methoxyindanone

5-methylindanone

5-methyl-6-methoxyindanone

5-methyl-7-chloroindanone

4-methoxy-7-chloroindanone

4-isopropyl-2,7-dimethylindanone

5,6,7-trichloroindanone

2-n-butylindanone

5-methylthioindanone

Scheme II has two mutually exclusive sub-schemes: Scheme IIA and SchemeIIB. Scheme IIA is used when R₃ is hydroxy and R₄ is hydrogen or whenthe two substituents form an oxo group. When R₃ is lower alkyl amino,Scheme IIB is employed.

Similar to Scheme I, in Scheme IIA the indenylacetic acid (k) in THF isallowed to react with oxalyichloride under reflux conditions to producethe acid chloride (p) (see reaction 18), whereupon the solvent isevaporated. In reaction 19, a 0° C. mixture of a benzyl hydroxylaminehydrochloride (q) and Et₃N is treated with a cold solution of the acidchloride in CH₂Cl₂ over a period of 45-60 minutes. The mixture is warmedto room temperature and stirred for one hour, and is treated with water.The resulting organic layer is washed with 1 N HCl and brine, is driedover magnesium sulfate and is evaporated. The crude product, aN-hydroxy-N-benzyl acetamide (r) is purified by crystallization or flashchromatography. This general procedure is taught by Hoffman et al., JOC1992, 57, 5700-5707.

The next step is the preparation of the N-mesyloxy amide (s) in reaction20, which is also taught by Hoffman et al., JOC 1992, 57, 5700-5707.Specifically, to a solution of the hydroxamic acid (r) in CH₂Cl₂ at 0°C. is added triethylamine. The mixture is stirred for 10-12 minutes, andmethanesulfonyl chloride is added dropwise. The mixture is stirred at 0°C. for two hours, is allowed to warm to room temperature, and is stirredfor another two hours. The organic layer is washed with water, 1 N HCl,and brine, and is dried over magnesium sulfate. After rotaryevaporation, the product(s) is usually purified by crystallization orflash chromatography.

The preparation of the N-benzyl-α-(hydroxy) amide (t) in reaction 21, isalso taught by Hoffman et al., JOC 1992, 57, 5700-5707 and Hoffman etal., JOC 1995, 60, 4121-4125. Specifically, to a solution of theN-(mesyloxy) amide (s) in CH₃CN/H₂O is added triethylamine in CH₃CN overa period of 6-12 hours. The mixture is stirred overnight. The solvent isremoved, and the residue is dissolved in ethyl acetate. The solution iswashed with water, 1 N HCl, and brine, and is dried over magnesiumsulfate. After rotary evaporation, the product (t) is usually purifiedby recrystallization.

Reaction 22 in Scheme IIA involves a condensation with certainaldehydes, which is described in Scheme III below, a scheme that iscommon to products made in accordance with Schemes I, IIA and IIB.

The final reaction 23 in Scheme IIA is the preparation of theN-benzyl-α-ketoamide (v), which involves the oxidation of a secondaryalcohol (u) to a ketone by e.g., a Pfitzner-Moffatt oxidation, whichselectively oxidizes the alcohol without oxidizing the Y group.Compounds (u) and (v) may be derivatized to obtain compounds with R₃ andR₄ groups as set forth in Formula I.

As explained above, Scheme II is employed when R₃ is lower alkyl amino.Similar to Scheme I, in Scheme IIB the indenylacetic acid (k) in THF isallowed to react with oxalylchloride under reflux conditions to producethe acid chloride (p) (see reaction 18), whereupon the solvent isevaporated. In reaction 24, a mixture of an alkyl hydroxylaminehydrochloride (i.e. HO—NHR where R is a lower alkyl, preferablyisopropyl) and Et₃N is treated at 0° C. with a cold solution of the acidchloride in CH₂Cl₂ over a period of 45-60 minutes. The mixture is warmedto room temperature and is stirred for one hour, and is diluted withwater. The resulting organic layer is washed with 1 N HCl and brine, isdried over magnesium sulfate and is evaporated. The crude product, aN-hydroxy-N-alkyl acetamide (w) is purified by crystallization or flashchromatography. This general procedure is also taught by Hoffman et al.,JOC 1992, 57, 5700-5707

The preparation of the N-mesyloxy amide (x) in reaction 25, which isalso taught by Hoffman et al., JOC 1992, 57, 5700-5707. Specifically, asolution of the hydroxamic acid (w) in CH₂Cl₂ at 0° C. is treated withtriethylamine, is stirred for 10-12 minutes, and is treated dropwisewith methanesulfonyl chloride. The mixture is stirred at 0° C. for twohours, is allowed to warm to room temperature, and is stirred foranother two hours. The resulting organic layer is washed with water, 1 NHCl, and brine, and is dried over magnesium sulfate. After rotaryevaporation, the product (x) is usually purified by crystallization orflash chromatography.

The preparation of the N-benzyl indenyl-α-loweralkylainino-acetamidecompound (y) in Scheme IIB as taught by Hoffman et al., JOC 1995, 60,4121-25 and J. Am. Chem Soc. 1993, 115, 5031-34, involves the reactionof the N-mesyloxy amide (x), with a benzylamine in CH₂Cl₂ at 0° C. isadded over a period of 30 minutes. The resulting solution is stirred at0° C. for one hour and at room temperature overnight. The solvent isremoved, and the residue is treated with 1 N NaOH. The extract withCH₂Cl₂ is washed with water and is dried over magnesium sulfate. Afterrotary evaporation, the product (y) is purified by flash chromatographyor crystallization.

Scheme III involves the condensation of the heterocycloaldehydes (i.e.,Y—CHO) with the indenyl amides to produce the final compounds of FormulaI. This condensation is employed, for example, in reaction 17 in SchemeI above and in reaction 22 in Scheme IIA. It is also used to convertcompound (y) in Scheme IIB to final compounds of Formula I.

In Scheme III, the amide (a′) from the above schemes, anN-heterocycloaldehyde (z), and sodium methoxide (1 M in methanol) arestirred at 60° C. under nitrogen for 24 hours. After cooling, thereaction mixture is poured into ice water. A solid is filtered off, iswashed with water, and is dried in vacuo. Recrystallization provides acompound of Formula I in Schemes I and IIB and the intermediate (u) inScheme IIA.

As has been pointed out above, it is preferable in the preparation ofmany types of the compounds of this invention, to use a nitrosubstituent on the benzene ring of the indanone nucleus and convert itlater to a desired substituent since by this route a great manysubstituents can be reached. This is done by reduction of the nitro tothe amino group followed by use of the Sandmeyer reaction to introducechlorine, bromine, cyano or xanthate in place of the amino. From thecyano derivatives, hydrolysis yields the carboxamide and carboxylicacid; other derivatives of the carboxy group such as the esters can thenbe prepared. The xanthates, by hydrolysis, yield the mercapto group thatmay be oxidized readily to the sulfonic acid or alkylated to analkylthio group that can then be oxidized to alkylsulfonyl groups. Thesereactions may be carried out either before or after the introduction ofthe 1-substituent.

The foregoing may be better understood from the following examples thatare presented for purposes of illustration and are not intended to limitthe scope of the invention. As used in the following examples, thereferences to substituents such as R₁, R₂, etc., refer to thecorresponding compounds and substituents in Formula I above.

EXAMPLE 1(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

(A) p-Fluoro-α-methylcinnamic Acid

p-Fluorobenzaldehyde (200 g, 1.61 mol), propionic anhydride (3.5 g, 2.42mol) and sodium propionate (155 g, 1.61 mol) are mixed in a one literthree-necked flask which had been flushed with nitrogen. The flask isheated gradually in an oil-bath to 140° C. After 20 hours, the flask iscooled to 100° C. and poured into 8 l of water. The precipitate isdissolved by adding potassium hydroxide (302 g) in 2 l of water. Theaqueous solution is extracted with ether, and the ether extracts arewashed with potassium hydroxide solution. The combined aqueous layersare filtered, are acidified with concentrated HCl, and are filtered. Thecollected solid, p-fluoro-α-methylcinnamic acid, is washed with water,and is dried and used as obtained.

(B) p-Fluoro-α-methylhydrocinnamic Acid

To p-fluoro-α-methylcinnamic acid (177.9 g, 0.987 mol) in 3.6 l ethanolis added 11.0 g of 5% Pd/C. The mixture is reduced at room temperatureunder a hydrogen pressure of 40 p.s.i. When hydrogen uptake ceases, thecatalyst is filtered off, and the solvent is evaporated in vacuo to givethe product, p-fluoro-α-methylhydrocinnamic acid, which was useddirectly in the next step.

(C) 6-Fluoro-2-methylindanone

To 932 g polyphosphoric acid at 70° C. (steam bath) is addedp-fluoro-α-methylhydrocinnamic acid (93.2 g, 0.5 mol) slowly withstirring. The temperature is gradually raised to 95° C., and the mixtureis kept at this temperature for 1 hour. The mixture is allowed to cooland is added to 2 l. of water. The aqueous suspension is extracted withether. The extract is washed twice with saturated sodium chloridesolution, 5% Na₂CO₃ solution, and water, and is dried, and isconcentrated on 200 g silica-gel; the slurry is added to a five poundsilica-gel column packed with 5% ether-petroleum ether. The column iseluted with 5-10% ether-petroleum ether, to give6-fluoro-2-methylindanone. Elution is followed by TLC.

(D) 5-Fluoro-2-methylindenyl-3-acetic Acid

A mixture of 6-fluoro-2-methylindanone (18.4 g, 0.112 mol), cyanoaceticacid (10.5 g, 0.123 mol), acetic acid (6.6 g), and ammonium acetate (1.7g) in dry toluene (15.5 ml) is refluxed with stirring for 21 hours, asthe liberated water is collected in a Dean Stark trap. The toluene isevaporated, and the residue is dissolved in 60 ml of hot ethanol and 14ml of 2.2 N aqueous potassium hydroxide solution. 22 g of 85% KOH in 150ml of water is added, and the mixture refluxed for 13 hours undernitrogen. The ethanol is removed under vacuum, and 500 ml water isadded. The aqueous solution is extracted well with ether, and is thenboiled with charcoal. The aqueous filtrate is acidified to pH 2 with 50%cold hydrochloric acid. The precipitate is dried and5-fluoro-2-methylindenyl-3-acetic acid (M.P. 164-166° C.) is obtained.

(E) 5-Fluoro-2-methylindenyl-3-acetyl Chloride

5-fluoro-2-methylindenyl-3-acetic acid (70 mmol) in THF (70 ml) isallowed to react with oxalylchloride (2 M in CH₂Cl₂; 35 ml; 70 mmol)under reflux conditions (24 hours). The solvent is evaporated to yieldthe title compound, which is used as such in the next step.

(F) 5-Fluoro-2-methyl-3-(N-benzyl-indenylacetamide

Benzylamine (5 mmol) is added slowly at room temperature to a solutionof 5-fluoro-2-methylindenyl-3-acetyl chloride (2.5 mmol.) in CH₂Cl₂ (10ml). The reaction mixture is refluxed overnight, and is extracted withaqueous HCl (10%), water, and aqueous NaHCO₃ (5%). The organic phase isdried (Na₂SO₄) and is evaporated to give the title compound, which isrecrystallized from CH₂Cl₂ to give the title compound as a white solid(m.p. 144° C).

(G)(Z)-5-Fluoro-2-methyl-(4-pyridinhlidene)-3-(N-benzyl-indenylacetamide

5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (3.38 mmol),4-pyridinecarboxaldehyde (4 mmol), sodium methoxide (1M NaOCH₃ inmethanol (30 ml)) are heated at 60° C. under nitrogen with stirring for24 hours. After cooling, the reaction mixture is poured into ice water(200 ml). A solid is filtered off, washed with water, and dried invacuo. Recrystallization from CH₃CN gives the title compound (m.p. 202°C.) as a yellow solid (R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H. R₆=H, R₇=H, n=1,m=1, Y=4-pyridinyl).

(H)(E)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

The mother liquor obtained from the CH₃CN recrystallization of 1G isrich on the geometrical isomer of 1G. The E-isomer can be obtained pureby repeated recrystallizations from CH₃CN.

EXAMPLE 2(Z)-5-Fluoro-2-methyl-(3-pyridinylidene)-3-(N-benzyl)-indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 3-pyridinecarboxaldehyde. Recrystallization from CH₃CN gives thetitle compound (m.p. 175° C.)(R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H, R₆=H,R₇=H, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 3(Z)-5-Fluoro-2-methyl-(2-pyridinylidene)-3-(N-benzyl-indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 2-pyridinecarboxaldehyde. Recrystallization from ethylacetate givesthe title compound (m.p. 218° C.)(R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H, R₆=H,R₇=H, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 4(Z)-5-Fluoro-2-methyl-(4-quinolipylidene)-3-(N-benzyl)-indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 4-quinolinecarboxaldehyde. Recrystallization from ethylacetategives the title compound (m.p. 239° C.)(R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H,R₆=H, R₇=H, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 5(Z)-5-Fluoro-2-methyl-(4,6-dimethyl-2-pyridinylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 4,6-dimethyl-2-pyridinecarboxaldehyde accordingto the procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4,6-dimethyl-2-pyridinyl).

EXAMPLE 6(Z)-5-Fluoro-2-methyl-(3-quinolinylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 3-quinolinecarboxaldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=3-quinolinyl)

EXAMPLE 7(Z)-5-Fluoro-2-methyl-(2-quinolinylidene-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 2-quinolinecarboxaldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=2-quinolinyl).

EXAMPLE 8(Z)-5-Fluoro-2-methyl-(Pyrazinylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyrazinealdehyde according to the procedure ofExample 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m−1, Y=pyrazinyl).

EXAMPLE 9(Z)-5-Fluoro-2-methyl-(3-pyridazinylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyridazine-3-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 10(Z)-5-Fluoro-2-methyl-(4-pyrimidinylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyrimidine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyrimidinyl).

EXAMPLE 11(Z)-5-Fluoro-2-methyl-(2-methyl-4-pyrimidinylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 2-methyl-pyrimidine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=2-methyl-4-pyrimidinyl).

EXAMPLE 12(Z)-5-Fluoro-2-methyl-(4-pyridazinylidene-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyridazine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 13(Z)-5-Fluoro-2-methyl-(1-methyl-3-indolylidene-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 1-methylindole-3-carboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=1-methyl-3-indolyl).

EXAMPLE 14(Z)-5-Fluoro-2-methyl-(1-acetyl-3-indolylidene)-3-(N-benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 1-acetyl-3-indolecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=1-acetyl-3-indolyl).

EXAMPLE 15(Z)-5-Fluoro-2-methyl-(4-pyrdinylidene)-3-(N-2-fluorobenzyl)-indenylacetamide

(A) 5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide

This compound is obtained from 5-fluoro-2-methylindenyl-3-acetylchloride (Example 1E) using the procedure of Example 1, Part F andreplacing benzylamine with 2-fluorobenzylamine.

(B)(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-2-fluorobenzy)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide is allowed toreact with 4-pryidinecarboxaldehyde according to the procedure ofExample 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 16(Z)-5-Fluoro-2-methyl-(3-pyridinylidene)-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 3-pryidinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 17(Z)-5-Fluoro-2-methyl-(2-pyridinylidene)-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 2-pyridinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=F, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 18(Z)-5-Fluoro-2-methyl-(4-quinolinlidene)-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 4-quinolinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=3-quinolinyl).

EXAMPLE 19(Z)-5-Fluoro-2-methyl-(3-pyrazinylidene)-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pyrazinealdehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyrazinyl).

EXAMPLE 20(Z)-5-Fluoro-2-methyl-(3-pyridazinylidene-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 3-pryidaziine-3-aldehyde according tothe procedure of Example 1, Part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆ =H, R₇=F, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 21(Z)-5-Fluoro-2-methyl-(3-pyrimidinylidene)-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pryimidine-4-aldehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyrimidinyl).

EXAMPLE 22(Z)-5-Fluoro-2-methyl-(4-pyridazylidene-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pryidazine-4-aldehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 23(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

(A) 5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide

5-Fluoro-2-methylindenyl-3-acetic acid (from Example 1D) (2.6 mmol) inDMA (2 ml) is allowed to react withn-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4 mmol)and S-2-amino-2-phenylethanol (3.5 mmol) at room temperature for twodays. The reaction mixture is added dropwise to stirred ice water (50ml). A white precipitate is filtered off, washed with water (5 ml), anddried in vacuo. Recrystallization from ethylacetate gives the desiredcompound.

(B)(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide frompart A is allowed to react with 4-pryidinecarboxaldehyde according tothe procedure of Example 1, Part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 24(Z)-5-Fluoro-2-methyl-(3-pyridinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 3-pryidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 25(Z)-5-Fluoro-2-methyl-(2-pyridinylidene)-3-(N-(S-α-hydroxymethyl)benzyl-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 2-pryidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 26(Z)-5-Fluoro-2-methyl-(4-quinolinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 4-quinolinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-quinolinyl).

EXAMPLE 27(Z)-5-Fluoro-2-methyl-(pyrazidinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryazidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=pyrazidinyl).

EXAMPLE 28(Z)-5-Fluoro-2-methyl-(3-pyridazinylidene)-3-(N-S-α-hydroxymethyl)benzyl-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryidazine-3-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 29(Z)-5-Fluoro-2-methyl-(4-pyrimidinylidene)-3-(N-(S-α-hydroxymethyl)benzyl-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryimidine-4-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-pyrimidinyl).

EXAMPLE 30(Z)-5-Fluoro-2-methyl-(4-pyridazinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryidazine-4-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 31rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)indenyl-α-hydroxyacetamide

(A) 5-Fluoro-2-methyl-3-(N-benzyl-N-hydroxy)-indenylacetamide

To a mixture of N-benzylhydroxylamine hydrochoride (12 mmol) and Et₃N(22 mmol) in CH₂Cl₂ (100 ml) at 0° C. is added a cold solution of5-Fluoro-2-methylindenyl-3-acetyl chloride (Example 1, Step E) (10 mmol)in CH₂Cl₂ (75 ml) over a period of 45-60 minutes. The mixture is warmedto room temperature and is stirred for 1 hour. The mixture is dilutedwith water (100 ml), and the organic layer is washed with HCl (2×25 ml)and brine (2×100 ml), dried (MgSO₄) and evaporated. The crude product ispurified with flash chromatography to give the title compound.

(B) 5-Fluoro-2-methyl-3-(N-benzyl-N-mesyloxy)-indenylacetamide

To a solution of5-Fluoro-2-methyl-3-(N-benzyl-N-hydroxy)-indenylacetamide (5 mmol) inCH₂Cl₂ (25 ml) at 0° C. is added triethylamine (5 mmol). The mixture isstirred for 10 minutes, and methanesulfonyl chloride (5.5 mmol) is addeddropwise. The solution is stirred at 0° C. for 2 hours, allowed to warmto room temperature, and stirred for another 2 hours. The organic layeris washed with water (2×20 ml), in HCl (15 ml), and brine (20 ml) anddried over MgSO₄. After rotary evaporation, the product is purified withflash chromatography to give the title compound.

(C) rac-5-Fluoro-2-methyl-3-(N-benzyl)-α-hydroxyindenylacetamide

To a solution of5-Fluoro-2-methyl-3-(N-benzyl-N-mesyloxy)-indenylacetamide (2 mmol) inCH₃CN/H₂O (12 ml. each) is added triethylamine (2.1 mmol) in CH₃CN (24ml) over a period of 6 hours. The mixture is stirred overnight. Thesolvent is removed, and the residue diluted with ethyl acetate (60 ml),washed with water (4×20 ml), in HCl (15 ml), and brine (20 ml) and driedover MgSO₄. After rotary evaporation, the product is purified byrecrystallization to give the title compound.

(D)rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-hydroxyacetamideis obtained fromrac-5-Fluoro-2-methyl-3-(N-benzyl)-α-hydroxyindenylacetamide using theprocedure of Example 1, Part G (R₁=F, R₂=CH₃, R₃=OH, R₄=H, R₅=H, R₆=H,R₇=H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 322-[(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl]-oxyacetamide

For Pfitzner-Moffatt oxidation, a solution ofrac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-hydroxyacetamide(1 mmol) in DMSO (5 ml) is treated with dicyclohexylcarbodiimide (3mmol). The mixture is stirred overnight, and the solvent is evaporated.The crude product is purified by flash chromatography to give the titlecompound (R₁=F, R₂=CH₃, R₃ and R₄ together form C═O, R₅=H, R₆=H, R₇=H,n=1, m=1, and Y=4-pyridinyl).

EXAMPLE 33rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-(2-propylamino)-acetamide

(A) 5-luoro-2-methyl-3-(N-2-propyl-N-hydroxy)-indenylacetamide isobtained from 5-fluoro-2-methylindenyl-3-acetyl chloride (Example 1,Step E) using the procedure of Example 31, Part A and replacingN-benzylhydroxylamine hydrochloride with N-2-propyl hydroxylaminehydrochloride.

(B) 5-Fluoro-2-methyl-3-(N-2-propyl-N-mesyloxy)-indenylacetamide isobtained according to the procedure of Example 31, Part B.

(C) rac-5-Fluoro-2-methyl-3-(N-benzyl)-α-(2-propylamino)-acetamide. To5-fluoro-2-methyl-3-(N-2-propyl-N-mesyloxy)-indenylacetamide (2 mmol) inCH₂Cl₂ (25 ml) at 0° C. is added benzylamine (4.4 mmol) in CH₂Cl₂ (15ml) over a period of 30 minutes. The resulting solution is stirred at 0°C. for 1 hour, and at room temperature overnight. The solvent isremoved, and the residue is treated with 1 N NaOH, and is extracted withCH₂Cl₂ (100 ml). The extract is washed with water (2×10 ml), and isdried over MgSO₄. After rotary evaporation, the product is purified byflash chromatography.

(D)rac-(Z)-5-fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl-indenyl-α-(2-propylamino)-acetamideis obtained fromrac-5-fluoro-2-methyl-3-(N-benzyl)-α-(2-propylamino)-acetamide using theprocedure of Example 1, Part G (R₁=F, R₂=CH₃, R₃=isopropylamino, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 34(Z)-6-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

(A) Ethyl-2-hydroxy-2-(p-methoxyphenyl)-1-methylpropionate

In a 500 ml. 3-necked flask is placed 36.2 g. (0.55 mole) of zinc dust,a 250 ml. addition funnel is charged with a solution of 80 ml. anhydrousbenzene, 20 ml. of anhydrous ether, 80 g. (0.58 mole) of p-anisaldehydeand 98 g. (0.55 mole) of ethyl-2-bromoproplonate. About 10 ml. of thesolution is added to the zinc dust with vigorous stirring, and themixture is warmed gently until an exothermic reaction commences. Theremainder is added dropwise at such a rate that the reaction mixturecontinues to reflux smoothly (ca. 30-35 min.). After addition iscompleted the mixture is placed in a water bath and refluxed for 30minutes. After cooling to 0°, 250 ml. of 10% sulfuric acid is added withvigorous stirring. The benzene layer is extracted twice with 50 ml.portions of 5% sulfuric acid and washed twice with 50 ml. portions ofwater. The combined aqueous acidic layers are extracted with 2×50 ml.ether. The combined etheral and benzene extracts are dried over sodiumsulfate. Evaporation of solvent and fractionation of the residue througha 6″ Vigreux column affords 89 g. (60%) of the product,ethyl-2-hydroxy-2-(p-methoxyphenyl)-1-methylpropionate, B.P. 165-160°(1.5 mm.).

(B) 6-Methoxy-2-methylindanone

By the method described in Vander Zanden, Rec. Trav. Chim., 68, 413(1949), the compound from part A is converted to6-methoxy-2-methylindanone.

Alternatively, the same compound can be obtained by addingα-methyl-β-(p-methoxylphenyl)propionic acid (15 g.) to 170 g. ofpolyphosphoric acid at 50° and heating the mixture at 83-90° for twohours. The syrup is poured into iced water. The mixture is stirred forone-half hour, and is extracted with ether (3×). The etheral solution iswashed with water (2×) and 5% NaHCO₃ (5×) until all acidic material hasbeen removed, and is dried over sodium sulfate. Evaporation of thesolution gives 9.1 g. of the indanone as a pale yellow oil.

(C)(Z)-6-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

In accordance with the procedures described in Example 1, parts D-G,this compound is obtained substituting 6-methoxy-2-methylindanone for6-fluoro-2-methylindanone in part D of Example 1.

EXAMPLE 35(Z)-5-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

(A) Ethyl 5-Methoxy-2-methyl-3-indenyl Acetate

A solution of 13.4 g of 6-methoxy-2-methylindanone and 21 g. of ethylbromoacetate in 45 ml. benzene is added over a period of five minutes to21 g. of zinc amalgam (prepared according to Org. Syn. Coll. Vol. 3) in110 ml. benzene and 40 ml. dry ether. A few crystals of iodine are addedto start the reaction, and the reaction mixture is maintained at refluxtemperature (ca. 65°) with external heating. At three-hour intervals,two batches of 10 g. zinc amalgam and 10 g. bromoester are added and themixture is then refluxed for 8 hours. After addition of 30 ml. ofethanol and 150 ml. of acetic acid, the mixture is poured into 700 ml.of 50% aqueous acetic acid. The organic layer is separated, and theaqueous layer is extracted twice with ether. The combined organic layersare washed thoroughly with water, ammonium hydroxide and water. Dryingover sodium sulfate, evaporation of solvent in vacuo followed by pumpingat 80° (bath temperature)(1-2 mm.) gives crudeethyl-(1-hydroxy-2-methyl-6-methoxy-indenyl) acetate (ca. 18 g.).

A mixture of the above crude hydroxyester, 20 g. of p-toluenesulfonicacid monohydrate and 20 g. of anhydrous calcium chloride in 250 ml.toluene is refluxed overnight. The solution is filtered, and the solidresidue is washed with toluene. The combined toluene solution is washedwith water, sodium bicarbonate, water and then dried over sodiumsulfate. After evaporation, the crude ethyl 5-methoxy-2-methyl-3-indenylacetate is chromatographed on acid-washed alumina, and the product iseluted with petroleum ether-ether (v./v. 50-100%) as a yellow oil (11.8g., 70%).

(B)(Z)-5-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingethyl-5-methoxy-2-methyl-3-indenyl acetate for5-fluoro-2-methylindenyl-3-acetic acid in Example 1, part E.

EXAMPLE 36(Z)-α-5-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylpropionamide

(A) α-(5-Methoxy-2-methyl-3-indenyl)propionic Acid

The procedure of Example 35, part (A) is followed using ethylα-bromopropionate in equivalent quantities in place of ethylbromoacetate used therein. There is obtained ethylα-(1-hydroxy-6-methoxy-2-methyl-1-indanyl)propionate, which isdehydrated to ethyl α-(5-methoxy-2-methyl-3-indenyl)propionate in thesame manner.

The above ester is saponified to giveα-(5-methoxy-2-methyl-3-indenyl)propionic acid.

(B)(Z)-α-5-Methoxy-2-methyl-(4-pyridinyl)-3-(N-benzyl)-α-methylindenylpropionamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingα-5-methoxy-2-methyl-3-indenyl)propionic acid for5-Fluoro-2-methylindenyl-3-acetic acid in Example 1, part E.

EXAMPLE 37 (Z)α-Fluoro-5-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)indenylacetamide

(A) Methyl-5-methoxy-2-methyl-3-indenyl-α-fluoro Acetate

A mixture of potassium fluoride (0.1 mole) andmethyl-5-methoxy-2-methyl-3-indenyl-α-tosyloxy acetate (0.05 mole) in200 ml. dimethylformamide is heated under nitrogen at the refluxtemperature for 2-4 hours. The reaction mixture is cooled, poured intoiced water and then extracted with ether. The ethereal solution iswashed with water, sodium bicarbonate and dried over sodium sulfate.Evaporation of the solvent and chromatography of the residue on anacid-washed alumina column (300 g.) using ether-petroleum ether (v./v.20-50%) as eluent give the product,methyl-5-methoxy-2-methyl-3-indenyl-α-fluoroacetate.

(B) (Z)α-Fluoro-5-methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)indenylacetamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingmethyl-5-methoxy-2-methyl-3-indenyl-α-fluoroacetate for5-Fluoro-2-methylindenyl-3-acetic acid in Example 1, part E.

For the introduction of the ═CH—Y part in Scheme III, any of theappropriate heterocyclic aldehydes may be used either directly in thebase-catalyzed condensation or in a Wittig reaction in an alternativeroute. The aldehydes that may be used are listed in Table 1 below:

TABLE 1 pyrrol-2-aldehyde* pyrimidine-2-aldehyde6-methylpyridine-2-aldehyde* 1-methylbenzimidazole-2-aldehydeisoquinoline-4-aldehyde 4-pyridinecarboxaldehyde*3-pyridinecarboxaldehyde* 2-pyridinecarboxaldehyde*4,6-dimethyl-2-pyridinecarboxaldehyde* 4-methyl-pyridinecarboxaldehyde*4-quinolinecarboxaldehyde* 3-quinolinecarboxaldehyde*2-quinolinecarboxaldehyde* 2-chloro-3-quinolinecarboxaldehyde*pyrazinealdehyde (Prepared as described by Rutner et al., JOC 1963, 28,1898-99) pyridazine-3-aldehyde (Prepared as described by Heinisch etal., Monatshefte Fuer Chemie 108, 213-224, 1977) pyrimidine-4-aldehyde(Prepared as described by Bredereck et al., Chem. Ber. 1964, 97,3407-17) 2-methyl-pyrimidine-4-aldehyde (Prepared as described byBredereck et al., Chem. Ber. 1964 97, 3407-17) pyridazine-4-aldehyde(Prepared as described by Heinisch et al., Monatshefte Fuer Chemie 104,1372-1382 (1973)) 1-methylindole-3-carboxaldehyde*1-acetyl-3-indolecarboxaldehyde* *Available from Aldrich

The aldehydes above can be used in the reaction schemes above incombination with various appropriate amines to produce compounds withthe scope of this invention. Examples of appropriate amines are thoselisted in Table 2 below:

TABLE 2 benzylamine 2,4-dimethoxybenzylamine 2-methoxybenzylamine2-fluorobenzylamine 4-dimethylaminobenzylamine 4-sulfonaminobenzylamine1-phenylethylamine (R-enantiomer) 2-amino-2-phenylethanol (S-enantiomer)2-phenylglycinonitrile (S-enantiomer)

EXAMPLE 38(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamideHydrochloride

(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamide(1396 g; MW 384.45; 3.63 mol) from Example 1 is dissolved at 45° C. inethanol (28 L). Aqueous HCl (12 M; 363 mL) is added stepwise. Thereaction mixture is heated under reflux for 1 hour, is allowed to coolto room temperature, then stored at −10° C. for 3 hours. The resultingsolid is filtered off, is washed with ether (2×1.5 L) and is air-driedovernight. Drying under vacuum at 70° C. for 3 days gives(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride with a melting point of 207-209° C. (R₁=F, R₂=CH₃, R₃=H,R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyridinyl·hydrochloride). Yield:1481 g (97%; 3.51 mol); MW: 420.91 g/mol.

¹H-NMR (DMSO-d₆): 2.18 (s, 3, ═C—CH₃); 3.54 (s, 2, ═CH₂CO); 4.28 (d, 2,NCH₂); 6.71 (m, 1, ar.); 7.17 (m, 8, ar.); 8.11 (d, 2, ar., AB system);8.85 (m, 1, NH); 8.95 (d, 2, ar., AB system); IR (KBr): 3432 NH; 1635C═O; 1598 C═C.

EXAMPLE 39(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamidep-methylbenzenesulfonate

(Z)-5-fluoro-2-methyl-(4-pyridylene)-3-(N-benzyl)indenylacetamide(MW=384.46 g/mol; 5.21 mmol; 2 g) from Example 1 is dissolved in ethanol(50 ml). Solid p-toluenesulfonic acid monohydrate (MW=190.22 g/mol; 5.21mmol; 991 mg) is added to the stirred solution. The reaction mixture isstirred for 12 hours at room temperature. The ethanol is evaporated inaspirator vacuum. The residue is dried in high vacuum to yield(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamidep-methylbenzenesulfonate as an orange-red powder.

As to identifying structurally additional PDE2 and PDE5 inhibitingcompounds besides those of Formula I that can be effectivetherapeutically for type I diabetes, one skilled in the art has a numberof useful model compounds disclosed herein (as well as their analogs)that can be used as the bases for computer modeling of additionalcompounds having the same conformations but different chemically. Forexample, software such as that sold by Molecular Simulations Inc.release of WebLab® ViewerPro™ includes molecular visualization andchemical communication capabilities. Such software includesfunctionality, including 3D visualization of known active compounds tovalidate sketched or imported chemical structures for accuracy. Inaddition, the software allows structures to be superimposed based onuser-defined features, and the user can measure distances, angles, ordihedrals.

In this situation, since the structures of active compounds aredisclosed above, one can apply cluster analysis and 2D and 3D similaritysearch techniques with such software to identify potential newadditional compounds that can then be screened and selected according tothe selection criteria of this invention. These software methods relyupon the principle that compounds, which look alike or have similarproperties, are more likely to have similar activity, which can beconfirmed using the PDE selection criterion of this invention.

Likewise, when such additional compounds are computer-modeled, many suchcompounds and variants thereof can be synthesized using knowncombinatorial chemistry techniques that are commonly used by those ofordinary skill in the pharmaceutical industry. Examples of a fewfor-hire combinatorial chemistry services include those offered by NewChemical Entities, Inc. of Bothell, Wash., Protogene Laboratories, inc.,of Palo Alto, Calif., Axys, Inc. of South San Francisco, Calif.,Nanosyn, Inc. of Tucson, Ariz., Trega, Inc. of San Diego, Calif., andRBI, Inc. of Natick, Mass. There are a number of other for-hirecompanies. A number of large pharmaceutical companies have similar, ifnot superior, in-house capabilities. In short, one skilled in the artcan readily produce many compounds for screening from which to selectpromising compounds for treatment of neoplasia having the attributes ofcompounds disclosed herein.

To further assist in identifying compounds that can be screened and thenselected using the criterion of this invention, knowing the binding ofselected compounds to PDE5 and PDE2 protein is of interest. By theprocedures discussed below, it is believed that preferable, desirablecompounds meeting the selection criteria of this invention bind to thecGMP catalytic regions of PDE2 and PDE5.

To establish this, a PDE5 sequence that does not include the catalyticdomain can be used. One way to produce such a sequence is to expressthat sequence as a fusion protein, preferably with glutiathioneS-transferase (“GST”), for reasons that will become apparent.

RT-PCR method is used to obtain the cGB domain of PDE5 with forward andreverse primers designed from bovine PDE5A cDNA sequence(McAllister-Lucas L. M. et al, J. Biol. Chem. 268, 22863-22873, 1993)and the selection among PDE 1-10 families. 5′-3′, Inc. kits for totalRNA followed by oligo (dT) column purification of mRNA are used withHT-29 cells. Forward primer (GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G,203-227) and reverse primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGC-TG,1664-1686) are used to synthesize the 1484 bp fragment coding for thephosphorylation site and both low and high affinity cGMP binding sitesof human PDE5A (203-1686 bp, cGB-pDE5). The synthesized cGB-pDE5nucleotide fragment codes for 494 amino acids with 97% similarity tobovine PDE5A. It is then cloned into pGEX-5X-3 glutathione-S-ransferase(GST) fusion vector (Pharmacia Biotech )with tac promoter, and EcoRI andXhoI cut sites. The fusion vector is then transfected into E. Coli BL21(DE3) bacteria (Invitrogen). The transfected BL21 bacteria is grown tolog phase, and then IPTG is added as an inducer. The induction iscarried at 20° C. for 24 hrs. The bacteria are harvested and lysed. Thesoluble cell lysate is incubated with GSH conjugated Sepharose 4B(GSH-Sepharose 4B). The GST-cGB-pDE5 fusion protein can bind to theGSH-Sepharose beads, and the other proteins are washed off from beadswith excessive cold PBS.

The expressed GST-cGB-pDE5 fusion protein is displayed on 7.5% SDS-PAGEgel as an 85 Kd protein. It is characterized by its cGMP binding andphosphorylation by protein kinases G and A. It displays two cGMP bindingsites, and the K_(d) is 1.6±0.2 μM, which is close to K_(d)=1.3 μM ofthe native bovine PDE5. The GST-cGB-PDE5 on GSH-conjugated sepharosebeads can be phosphorylated in vitro by cGMP-dependent protein kinaseand cAMP-dependent protein kinase A. The K_(m) of GST-cGB-pDE5phosphorylation by PKG is 2.7 μM and Vmax is 2.8 μM, while the K_(m) ofBPDEtide phosphorylation is 68 μM. The phosphorylation by PKG showsmolecular phosphate incorporated into GST-cGB-pDE5 protein on aone-to-one ratio.

A cGMP binding assay for compounds of interest (Francis S. H. et al, J.Biol. Chem. 255, 620-626, 1980) is done in a total volume of 100 μLcontaining 5 mM sodium phosphate buffer (pH=6.8), 1 mM EDTA, 0.25 mg/mLBSA, H³-cGMP (2 μM, NEN) and the GST-cGB-PDE5 fusion protein (30 pg/assay). Each compound to be tested is added at the same time as ³H-cGMPsubstrate, and the mixture is incubated at 22° C. for 1 hour. Then, themixture is transferred to Brandel MB-24 cell harvester with GF/B as thefilter membrane followed by 2 washes with 10 mL of cold 5 mM potassiumbuffer(pH 6.8). The membranes are then cut out and transferred toscintillation vials followed by the addition of 1 mL of H₂O and 6 mL ofReady Safe™ liquid scintillation cocktail to each vial. The vials arecounted on a Beckman LS 6500 scintillation counter.

For calculation, blank samples are prepared by boiling the bindingprotein for 5 minutes, and the binding counts are <1% when compare tounboiled protein. The quenching by filter membrane or other debris arealso calibrated.

PDE5 inhibitors, sulindac sulfide, exisulind, E4021 and zaprinast, andcyclic nucleotide analogs, cAMP, cyclic IMP, 8-bromo-cGMP, cyclic UMP,cyclic CMU, 8-bromo-cAMP, 2′-O-butyl-cGMP and 2′-O-butyl-cAMP wereselected to test whether they could competitively bind to the cGMPbinding sites of the GST-cGB-pDE5 protein. cGMP specifically bound toGST-cGB-PDE5 protein. Cyclic AMP, cUMP, cCMP, 8-bromo-cAMP,2′-O-butyl-cAMP and 2′-O-butyl-cGMP did not compete with cGMP inbinding. Cyclic IMP and 8-bromo-cGMP at high concentration (100 μM) canpartially compete with cGMP (2 μM) binding. None of the PDE5 inhibitorsshowed any competition with cGMP in binding of GST-cGB-PDE5. Therefore,they do not bind to the cGMP binding sites of PDE5.

However, Compound 38 does competitively (with cGMP) bind to PDE 5. Giventhat Compound 38 does not bind to the cGMP-binding site of PDE5, thefact that there is competitive binding between Compound 38 and cGMP atall means that desirable compounds such as Compound 38 bind to the cGMPcatalyic site on PDE5, information that is readily obtainable by oneskilled in the art (with conventional competitive binding experiments)but which can assist one skilled in the art more readily to model othercompounds. Thus, with the chemical structures of desirable compoundspresented herein and the cGMP binding site information, one skilled inthe art can model, identify and select (using the selection criteria ofthis invention) other chemical compounds for use as therapeutics.

Examples of compounds that inhibit PDE2 and PDE5 (with insubstantial COXinhibition) include exisulind and compounds disclosed in U.S. Pat. Nos.5,965,619 and 6,063,818 which are incorporated herein by reference.

BIOLOGICAL EFFECTS

(A) Cyclooxygenase (COX) Inhibition

COX catalyzes the formation of prostaglandins and thromboxane by theoxidative metabolism of arachidonic acid. The compound of Example 1 ofthis invention, as well as a positive control, (sulindac sulfide) wereevaluated to determine whether they inhibited purified cyclooxygenaseType I (see Table 1 below).

The compounds of this invention were evaluated for inhibitory effects onpurified COX. The COX was purified from ram seminal vesicles, asdescribed by Boopathy, R. and Balasubramanian, J., 239:371-377, 1988.COX activity was assayed as described by Evans, A. T., et al., “Actionsof Cannabis Constituents on Enzymes Of Arachidonate MetabolismAnti-Inflammatory Potential,” Biochem. Pharmacol., 36:2035-2037, 1987.Briefly, purified COX was incubated with arachidonic acid (100 μM) for2.0 min at 37° C. in the presence or absence of test compounds. Theassay was terminated by the addition of TCA, and COX activity wasdetermined by absorbance at 530 nm.

TABLE 1 COX I EXAMPLE % Inhibition(100 μM) Sulindac sulfide 86 1 <25

The advantage of very low COX inhibition is that compounds of thisinvention can be administered to patients without the side effectsnormally associated with COX inhibition.

(B) cGMP PDE Inhibition

Compounds of this invention are also PDE2 and PDE5 inhibitors as taughtin part U.S. patent application Ser. No. 09/046,739 filed Mar. 24, 1998.Compounds can be tested for inhibitory effect on phosphodiesteraseactivity using either the enzyme isolated from any tumor cell line suchas HT-29 or SW-480. Phosphodiesterase activity can be determined usingmethods known in the art, such as a method using radioactive ³H cyclicGMP (cGMP)(cyclic 3′,5′-guanosine monophosphate) as the substrate forPDE5 enzyme. (Thompson, W. J., Teraski, W. L., Epstein, P. M., Strada,S. J., Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which isincorporated herein by reference). In brief, a solution of definedsubstrate ³H-cGMP specific activity (0.2 μM; 100,000 cpm; containing 40mM Tris-HCl (pH 8.0), 5 mM MgCl₂ and 1 mg/ml BSA) is mixed with the drugto be tested in a total volume of 400 μl. The mixture is incubated at30° C. for 10 minutes with partially purified cGMP-specific PDE isolatedfrom HT-29 cells. Reactions are terminated, for example, by boiling thereaction mixture for 75 seconds. After cooling on ice, 100 μl of 0.5mg/ml snake venom (O. Hannah venom available from Sigmna) is added andincubated for 10 min at 30° C. This reaction is then terminated by theaddition of an alcohol, e.g. 1 ml of 100% methanol. Assay samples areapplied to a anion chromatography column (1 ml Dowex, from Aldrich) andwashed with 1 ml of 100% methanol. The amount of radioactivity in thebreakthrough and the wash from the columns in then measured with ascintillation counter. The degree of PDE5 inhibition is determined bycalculating the amount of radioactivity in drug-treated reactions andcomparing against a control sample (a reaction mixture lacking thetested compound).

Using such protocols, the compound of Example 1 had an IC₅₀ value forPDE5 inhibition of 0.68 μM. Using similar protocols, the compound ofExample 38 (“Compound 38”) had an IC₅₀ value for PDE2 of 14 μM, an IC₅₀value for PDE5 of 4 μM, an IC₅₀ value for PDE1 of 3 μM, and an IC₅₀value for PDE4 of 6 μM.

(C) Safety Assessment in Mammals

As one skilled in the art will recognize from the data presented below,Compound 38 can safely be given to animals at doses far beyond thetolerable (and in many cases toxic) doses of non-insulin type I diabetestherapies. For example, in an acute toxicity study in rats, single oraldoses of Compound 38 administered (in a 0.5% carboxymethylcellulosevehicle) at doses up to and including 2000 mg/kg resulted in noobservable signs of toxicity. At 2000 mg/kg, body weight gains wereslightly reduced. A single dose of 1000 mg/kg administeredintraperitoneally resulted in reduced body weight gain, with mesentericadhesions seen in some animals from this group at necropsy.

In dogs, the administration of Compound 38 in capsules at 1000 mg/kgresulted in no signs of toxicity to the single group of two male and twofemale dogs. Due to the nature of Compound 38 capsules, this dosenecessitated the use of at least 13 capsules to each animal, which wasjudged to be the maximum number without subjecting the animals tostress. Therefore, these dogs were subsequently administered sevenconsecutive doses of 1000 mg/kg/day. At no time in either dosing phasewere any obvious signs of drug-related effects observed.

Thus, on a single-dose basis, Compound 38 is not acutely toxic. Based onthe findings of these studies, the oral LD₅₀ of Compound 38 wasconsidered to be greater than 1000 mg/kg in dogs and 2000 mg/kg in rats,and the intraperitoneal LD₅₀ was considered to be greater than 1000mg/kg in rats.

A seven-day dose-range finding study in rats, where Compound 38 wasevaluated by administering it at doses of 0, 50, 500 or 2000 mg/kg/dayresulting in no observable signs of toxicity at 50 mg/kg/day. At 500mg/kg/day, treatment-related effects were limited to an increase inabsolute and relative liver weights in female rats. At 2000 mg/kg/day,effects included labored breathing and/or abnormal respiratory sounds,decreased weights gains and food consumption in male rats, and increasedliver weights in female rats. No hematological or blood chemistrychanges nor any microscopic pathology changes, were seen at any doselevel.

A 28-day study in rats was also carried out at 0, 50, 500 and 2000mg/kg/day. There were no abnormal clinical observations attributed toCompound 38, and body weight changes, ophthalmoscopic examinations,hematological and blood chemistry values and urinalysis examinationswere unremarkable. No macroscopic tissue changes were seen at necropsy.Organ weight data revealed statistically significant increase in liverweights at 2000 mg/kg/day, and statistically significant increases inthyroid weights for the 2000 mg/kg/day group. The slight liver andthyriod increases at the lower doses were not statistically significant.Histopathological evaluation of tissues indicated the presence of tracesof follicular cell hypertrophy, increased numbers of mitotic figures(suggestive of possible cell proliferation) in the thyroid gland andmild centrilobular hypertrophy in the liver. These changes weregenerally limited to a small number of animals at the 2000 mg/kg/daydose, although one female at 500 mg/kg/day had increased mitotic figuresin the thyroid gland. The findings in the liver may be indicative of avery mild stimulation of liver microsomal enzymes, resulting inincreased metabolism of thyroid hormones, which in turn resulted inthyroid stimulation.

A long-term safety assessment study was conducted in rats to investigateCompound 38 at 50, 200 and 500 mg/kg/day following repeated oral dosingfor 91 consecutive days. Orally administered Compound 38 did not produceany major toxicological effects in rats. The only finding was adose-related trend to increased liver and thyroid/parathyroid weightsnoted in males and females at 200 and 500 mg/kg/day. Microscopically,slight hepatocellular hypertrophy at 200 and 500 mg/kg/day groups,follicular cell hypertrophy at 500 mg/kg/day and increase inaccumulation of hyalin droplets in the kidneys at 200 and 500 mg/kg/daygroup. However, no changes in clinical biochemistry and hematology wereevident. These changes were not associated with any gross clinicalabnormality.

Dogs were also dosed orally with Compound 38 at 50, 150 and 300mg/kg/day for 91 consecutive days. There were no toxicological effectsin the dog following 91 days of dosing. Orange discoloration of thefeces (same color as Compound 38) was seen in the 150 and 300 mg/kg/daygroups. This finding suggested that most of Compound 38 was beingeliminated via the feces. Slightly lowered body weights were noted inthe highest dose group. This dose was also associated with increasedliver weights. However, there were no microscopic alterations to supportthe increase in liver weight. Therefore, we concluded that Compound 38is well tolerated in the dog.

Finally as to safety, in a single, escalating dose human clinical trial,patients, human safety study in which the drug was taken orally,Compound 38 produced no significant side effects at any dose (i.e., 50mg BID, 100 mg BID, 200 mg BID and 400 mg BID).—doses above the levelbelieved to be therapeutic for human patients. One skilled in the artshould recognize that any of the side effects observed in these safetystudies occurred at very high doses, in excess of recommended humandoses and are extremely minimal compared to what one would expect atsimilar doses of other proposed therapies.

(D) Efficacy for Type I Diabetes

i. Macrophage Involvement

As mentioned above, macrophages have been implicated in the progressionof Type I diabetes (see Jansen et al., Diabetes Vol. 43, No. 5 pp.667-675 (1994)). Specifically, macrophages have been found to invadepancreatic tissue including the islets and their surrounding tissue inmany animal models of diabetes including the BB/E rat and NOD mouse (seeWalker et al., Diabetes, Vol. 37 No. 9 pp. 1301-04 (1988) and Jun etal., Diabetes, Vol. 48, No. 1, pp. 34-42 (1999)). The invading activatedmacrophages play critical roles in the pathological events of IDDMprogression.

As demonstrated below, we found that macrophages contain PDE2 and PDE5,and the inhibition of PDE2 particularly with PDE5 inhibition leads toapoptosis of macrophage cells. We believe the administration of a PDE2inhibitor can treat the progression of Type I diabetes, particularlywhen PDE5 is also inhibited.

ii. PDE2 and PDE5 MRNA Levels in Treated and Untreated U937 Cells byRT-pCR

The U937 monocyte cell line was derived from a histocytic lymphoma andcan be driven to differentiate into an ‘activated macrophage like’ stateby treatment with 5 nM phorbal ester (TPA). Treated U937 cells becomeadherent, increase their cytoplasmic volume and expressmacrophage-specific cell surface markers. The presence and level of PDE2and 5 MRNA in both differentiated and non-differentiated U937 cells wasconfumed by performing RT-pCR experiments on total RNA.

U937 cells (from ATCC Rockville, Md.) were grown in RPMI mediasupplemented with 5% FCS, glutamine, antibiotic/antimycotic and sodiumpyruvate. Total RNA was isolated from two U937 cultures, one treatedwith 5 nM TPA for 48 hours and one grown in normal media as listedabove, using the Rouche High Pure RNA Isolation Kit (cat #1 828 665) asper manufacturers protocol. cDNA was then synthesized from the total RNAusing GibcoBRL SuperscriptII (Cat #18064-022) reverse transcriptase asper manufacturers protocol. The resulting cDNA was used as a templatefor RT-PCR reactions using primer sets specific for PDE2 (forward:CCCAAAGTGGAGACTGTCTACACCTAC, reverse: CCGGTTGTCTTCCAGCGTGTC) or PDE5(forward: GGGACTTTACCTTCTCATAC, reverse: GTGACATCCAAATGACTAGA). mRNA forPDE2 and 5 were both present in the untreated U937 cells. Upon treatmentwith TPA, the relative amounts of PDE2 mRNA increased 5 fold. Therefore,U937 cells treated with TPA and driven to differentiate into anactivated macrophage like state have elevated levels of PDE2 mRNA (seeFIG. 1).

iii. Confirmation of PDE2 and PDE5 Protein Within U937 Cells by IndirectImmunofluorescence

The presence of PDE2 and PDE5 protein within U937 cells was confirmed byindirect immunofluorescence (IIF). U937 cells were cultured as above.Two U937 cultures, one grown in the presence of 5 nM TPA for 48 hoursand one grown in normal media were processed. All cultures werecollected by centrifugation (Shandon Cytospin, 2 minutes @600 rpm) ontopoly-L lysine-coated slides and immediately fixed in fresh 3%paraformaldehyde buffered in PBS for 10 minutes. Adherent cultures weregrown on coverslips and fixed as above. Cells were permeablized in 0.2%Triton-100 for 2 minutes. Slides were blocked with blocking buffer (5%goat serum, 5% glycerol, 1% gelatin from cold water fish skin and 0.04%NaN₃ in PBS) for 1 hour at room temperature.

Slides were then incubated for 1 hour at 37° C. in a humid chamber withantibodies specific for PDE2 (generated in a sheep against the peptideTLAFQKEQKLKCECQA) or PDE5 (generated in sheep against the peptideCAQLYETSLLENKRNQV). The PDE5 antibody was used at a dilution of 1:200and the PDE2 antibody was used at a dilution of 1:100. All dilutionswere performed in blocking buffer. Slides were then washed 2× for 10minutes each in PBS and then incubated with a Cy3 conjugated secondaryantibody (Jackson ImmunoResearch laboratories, Inc. Cat. #713-166-147)diluted 1:1000 in blocking buffer, for 1 hour at 37° C. in a humidchamber. Slides were then washed 2× for 10 minutes each in PBS andcounterstained with DAPI (5 ng/ml) and mounted in VectaShield. Digitalimages were then obtained using a SPOT-2 camera and an Olympus IX-70fluorescent microscope. Both PDE2 and PDE5 are present in the cytoplasmof U937 cells. There is an increase in the level of both PDE2 and PDE5in TPA-treated U937 cells. These increased protein levels are seen indiscrete perinuclear foci (see FIGS. 2 through 5).

iv. Cyclic GMP Hydrolysis Within U937 Cells

cGMP-hydrolytic activity in TPA-treated and untreated U937 cells wasdetermined by performing a permeablized cell assay and direct analysisof enzyme activity in protein lysates. Both procedures achieved similarresults, namely, elevated activity in the treated cells compared tountreated cells.

The cGMP hydrolysis levels in permeablized U937 cells was performed bywashing the cells for 5 minutes with DMEM followed by cold PBS. Cellswere then placed on ice in 700 μl ice cold Tris-HCL buffer (20 mM; pH7.4) containing MgCl₂ (5 mM) 0.5% Triton X-100, and protein inhibitors(10 mM bezamidine, 10 μM TLDK, 2000 U/ml aprotinin, 2 μM leupeptin, 2 μMpepstatin A). The reaction was initiated by the addition of 100 pl of0.5 mg/ml snake venom and 0.25 μM cGMP or cAMP along with [³H]cGMP or[³H]cAMP, respectively. After incubating for 30 minutes at 30° C. thereactions were terminated by the addition of 1.8 ml methanol. Theextract was then applied to a 1 ml Dowex anion exchange column to removeunreacted substrate. The eluant was collected and counted in 6 mlscintillation fluid. As shown in FIG. 6, U937 cell cGMP hydrolysislevels elevate when the cells are driven into an activatedmacrophage-like state upon treatment with TPA, as compared tounactivated, untreated cells.

cGMP hydrolysis levels in protein lysates extracted from TPA-treated anduntreated U937 cells were also analyzed as follows. Cells wereresuspended in 20 mM TRIS-HCl, 5 mM MgCl2, 0.5% Triton X-100, 0.1 mMEDTA, 10 mM benzamidine, 10 μM TLCK, 20 nM aprotinin, 2 μM leupeptin, 2μM pepstatin A, pH 8.0 were added. The cells were homogenized using aglass tissue grinder and teflon pestle. Samples were ultracentrifuged at100,000×g for 1 hr at 0° C. Supernatants were assayed at 0.25 μM cGMPusing the method from Thompson, W. J. et. al. Adv. Cyclic NucleotideRes., 10: 69-92, 1979. Again, the level of cGMP hydrolytic activityincreased upon TPA treatment/activation, compared with notreatment/unactivation (see FIG. 7). Both of these experimentscorroborate the results of our experiments above that show that bothcGMP PDE2 and PDE5 protein levels increase in U937 cells treated withTPA.

v. Apoptosis Induction of U937 Cells by Compound 38

U937 cells were cultured, as described above, with and without treatmentwith 5 nM TPA for 24 hours at which time the cultures were treatedeither with 1 μM Compound 38 or vehicle (DMSO) alone for an additional24 hours. Adherent cells were dislodged by treatment with trypsin EDTAfor 5 minutes at 37° C. Cells were then processed for IIF as describedabove, except that an antibody specific for active caspase 3 was used(as per manufacturer's protocol) instead of antibodies to PDE2 or 5(Promega Cat. #G7481). The anti-active caspase 3 antibody was diluted1:200 in blocking buffer and processed according to the manufacturer'sprotocol. The resulting slides were observed under a fluorescentmicroscope and a digital images were obtained. FIG. 8 shows U937 cellstreated with 1 μM compound 38 undergoing apoptosis as reflected by thepresence of active caspase 3 (red signal). Image of control (vehicleonly) U937 cells reveals only low, background levels of apoptosis (FIG.9).

The level of apoptosis in U937 cells was quantified by scoring 500consecutive cells for the presence of active caspase 3. These resultsare summarized in the following table.

Cell Number of Percentage of type TPA treatment Compound 38 apoptoticcells apoptotic cells U937 6/500 1.2% U937 1 μM, 24 hrs 375/500 75% U9375 nM, 16 hrs 59/500 11.8% U937 5 nM, 16 hrs 1 μM, 24 hrs 392/500 78%

Therefore, compound 38 causes the induction of apoptosis in thedifferentiated and non-differentiated U937 cell line.

vi. Treatment of U937 Cells With Either Sildenafil (PDE5-SpecificInhibitor) or Rolipram (PDE4-Specific Inhibitor) Does Not InduceApoptosis.

The activity of specific PDE inhibitors contrast with the activity ofcompound 38 in U937 cells. By “specific” in this context, we mean theother PDE inhibitors that inhibited one PDE primarily, but not severalPDEs (e.g., inhibiting PDE2 and PDE5 at roughly the same concentration).An example is sildenafil, which primarily inhibits PDE5, and only atmuch higher concentrations may only marginally inhibit other PDEs.Another example is rolipram (PDE4-specific).

U937 cells were incubated in the presence of 0.3 nM sildenafil or 0.5 uMrolipram for 24 hours using the culture conditions described above. Thecells were harvested and processed for IIF as described above using anantibody that specifically recognizes active caspase 3. Digital imagesare shown in FIGS. 10 and 11. No increase in the levels of apoptosiscompared to normal background was observed. Therefore, the inhibition ofonly PDE4 or PDE5 alone (i.e. without the inhibition of PDE2) is notsufficient to induce apoptosis in U937 cells.

vii. Compound 38 Decreases TNF Alpha Levels in U937 Media

One function of macrophages is to modulate the activity of otherinflammatory cells through various cytokine molecules. We thereforetested the effect of compound 38 on the ability of U937 cells to produceand secrete tumor necrosis factor-α (TNF-α). This was done by performingan immunoassay on the cell culture media taken from differentiated U937cells (TPA treated) grown in the presence or absence of compound 38.

TNF-α levels in the cell culture media were determined by using theTNF-α Immunoassay from R&D Systems (Cat. #DTA50) according to themanufacturer's protocol. As shown in FIG. 12, Compound 38 treatmentsignificantly reduced the level of TNF-α secreted by TPA-induced U937cells.

viii Phosphodiesterase and Macrophage Involvement In A Type I DiabetesAnimal Model

BBDP/Wor and BBDR/Wor Rats

The BB/Wor rat develops spontaneous and viral-induced syndromes ofauto-immune diabetes mellitus, and reportedly is the best rat model ofhuman Type 1 diabetes. Salient features include: genetic predisposition,abrupt onset of insulin dependent, ketosis-prone diabetes, andauto-immune destruction of pancreatic β-cells. Since the firstdescription of the syndrome in 1977, more than one thousand papers havebeen published by laboratories throughout the world. Support for theimmune pathogenesis of diabetes in the BB/Wor rat is derived from thefollowing: lymphocytic insulitis prior to and during the acute onset ofhyperglycemia; selective destruction of the pancreatic β-cells withsparing of the other islet cells; prevention and/or amelioration ofβ-cell destruction by immune suppressive agents directed against T cellsand macrophages, and measures which correct the effects of geneticallyinduced T cell lymphopenia; adoptive transfer insulitis and diabetes tonaive recipients with Conconavalin-A (CON-A) activated acute diabeticspleen cells. Other features of the BB/Wor rat which identify it as thebest rat model of human IDDM include their inbred status (>75generations of sib matings) and high (80-95%) incidence of diabetesamong both genders, and an average age onset of diabetes at 70days.<O:P</O:P

Pancreatic tissue was collected from three BBDP/Wor female ratscharacterized as having diabetes mellitus (type I diabetes). Animalswere randomized into three groups (n=1) and were characterizedphenotypically into prone- (early stage disease), acute-(mid-stagedisease) and chronic- (late-stage disease, insulin-dependent) stage typeI diabetes. In addition, three age-matched resistant controls (BBDR/Wor)were included into the study.

Phosphodiesterase Labeling Procedure

The BBDP/Wor rat pancreatic tissue specimens were fixed in 10% bufferedformalin, paraffin embedded, sectioned at 5 μm and stored at roomtemperature in a slide box until use. Sections were then dewaxed for 2hours at 60° C. and deparaffinized in xylene, three incubations, twominutes each. Sections were rehydrated through an ethanol series atthree different percentages (100, 95 and 70%), twice each for twominutes, followed by five minutes of rinsing in phosphate bufferedsaline. Endogenous peroxidase activity was blocked by incubatingpancreatic sections in fresh 0.3% hydrogen peroxide at room temperaturefor 30 minutes. Sections were washed in phosphate buffered saline for 5minutes, then blocked with blocking buffer (5% goat serum, 5% glycerol,1% gelatin from cold water fish skin and 0.04% NaN₃ in phosphatebuffered saline) for one hour. Tissue sections were incubated withprimary antibody diluted in blocking buffer (affinity purified sheepanti-human PDE2(1), diluted 1:1500 and affinity purified sheepanti-human PDE5(1), diluted 1:2000) and were incubated overnight at 4°C. in a humid chamber. Sections were washed three times for five minuteseach in phosphate buffered saline. Sections were incubated with asecondary antibody (diluted donkey anti-sheep biotin conjugatedimmunoabsorbed antibody 1:2500) in blocking buffer, JacksonlmmunoResearch Catalog #713-065-147. Sections were then incubated atroom temperature for 30 minutes, washed three times in phosphatebuffered saline for 5 minutes. Diluted Vector ABC reagents were used asaccording to manufacture's protocol (vector Catalog #6106) and wereincubated for 30 minutes at room temperature. Washed in phosphatebuffered saline for 5 minutes. A DAB substrate kit was used permanufacture's protocol (Vector Catalog #SK-4100). Slides containingtissue were rinsed with distilled water and immediately immerse inMeyers hematoxylin for 1-5 minutes. Excess hematoxylin was rinsed offwith distilled water. Slides were dipped 10 times in 2% glacial aceticacid solution followed by 10 dips in distilled water. Slides wereincubated in bluing solution (15 mM ammonium water) 15 dips followed by10 dips in water and dehydrated through an ethanol series. The labelledslides were then mounted in Permount (Sigma).

Macrophage Labeling Procedure

Rat tissue specimens were fixed in 10% buffered formalin, paraffinembedded, sectioned at 5 μm and stored at room temperature in a slidebox until use. Sections were then dewaxed for 2 hours at 60° C. anddeparaffinized in xylene, three incubations, two minutes each. Sectionswere rehydrated through an ethanol series at three differentpercentages, twice each for two minutes, followed by five minutes ofrinsing in phosphate buffered saline. Endogenous peroxidase activity wasblocked by incubating pancreatic sections in fresh 0.3% hydrogenperoxide at room temperature for 30 minutes. Sections were washed inphosphate buffered saline for 5 minutes, then blocked with blockingbuffer (5% goat serum, 5% glycerol, 1% gelatin from cold water fish skinand 0.04% NaN₃ in phosphate buffered saline) for one hour. Tissuesections were incubated with primary antibody diluted in blocking buffer(anti-macrophage antibody, diluted 1:1000, Serotec Catalog #MCA341R.)and were incubated overnight at 4° C. in a humid chamber. Sections werewashed three times for five minutes each in phosphate buffered saline.Sections were incubated with a secondary antibody (biotin anti-mouseantibody 1:2500) in blocking buffer. Sections were then incubated atroom temperature for 30 minutes, washed three times in phosphatebuffered saline for 5 minutes. Diluted Vector ABC reagents were used asaccording to manufacture's protocol (vector Catalog #6106) and wereincubated for 30 minutes at room temperature. Washed in phosphatebuffered saline for 5 minutes. A DAB substrate kit was used permanufacture's protocol (Vector Catalog #SK-4100). Slides containingtissue were rinsed with distilled water and immediately immerse inMeyers hematoxylin for 1-5 minutes. Excess hematoxylin was rinsed offwith distilled water. Slides were dipped 10 times in 2% glacial aceticacid solution followed by 10 dips in distilled water. Slides wereincubated in bluing solution (15 mM ammonium water) 15 dips followed by10 dips in water and dehydrated through an ethanol series. The labelledslides were then mounted in Permount (Sigma).

Quantitative PDE2 and PDE5 Immunohistochemistry

Rat pancreatic tissues on microscope slides were analyzed by theAutomated Cellular Imaging System (Chromavision, Capistrano, Calif.)using the generic DAB application (rev. A). Each tissue specimen wasthoroughly scanned and 10 islet regions were examined and areas ofnecrosis were avoided. Percentage of cells with positive signal for PDE2and PDE5 was computed. See table below for generated values (Table Iunder results section).

Quantitative Macrophage Immunohistochemistry Procedure

Beta islet regions of rat pancreatic tissue sections weremicroscopically observed with an Olympus light microscope (model #BX40),Olympus Optical CO., LTD, Japan. Objective lens used was 40×magnification. For each tissue section, approximately six beta isletregions were viewed for evidence of macrophages, and the total number ofmacrophages per tissue sample with positive signal was computed. Areasof necrosis were avoided. See table II below under “results” forgenerated values.

Results

Microscopically, the prone BBDP/Wor rat pancreatic tissue displayed nopositively stained macrophages in the islet region, however, earlyrecruitment of activated macrophages were evident in vasodilatedcapillaries as photographed in FIG. 13a. Its prone-resistant,aged-matched control showed no pancreatic tissue-laden macrophages (FIG.13b).

In contrast, following microscopic assessment, the highest number oftissue-activated macrophages was observed in the acute BBDP/Wor ratpancreas (FIG. 14a), and no macrophages were noted in the acuteresistant, age-matched control (FIG. 14b).

The chronic-staged BBDP/Wor pancreas showed a reduced number oftissue-activated macrophages (FIG. 15a), compared to the acute model.The chronic aged-matched control (chronic BBDR/Wor) showed no evidenceof macrophages in its pancreatic tissue section (FIG. 15b). FIG. 16denotes a bar graph of the total number of pancreatic tissue-activatedmacrophages for all groups/stages.

Rodent tissue-activated macrophage counts were statistically computedfrom islet pancreatic regions and results were graphed using GraphPadPrism® program. Paired t test was used to compare each BBDP/Wor Type-1stage to its appropriate control. There was a statistically significantdifference of the total number of tissue-activated macrophages betweenthe acute and chronic BBDP/Wor and their aged-matched controls (p=0.0003and p=0.02, respectively). There was no statistically significantdifference of the total number of tissue-activated macrophages betweenthe prone BBDP/Wor and the aged-matched control (p=0.796). Tukey'sMultiple Comparison Test was used to compared the total acute BBDP/Wortissue-activated macrophage count to all other groups and there was astatistically significant difference (p value less than 0.001).

With these results, one aspect of this invention is a medical therapythat involves the administration of the PDE2 inhibitor (preferably alsoa PDE5 inhibitor) at the stage of Type I diabetes development whenmacrophage invasion is occurring. Macrophages release cytokines that canrecruit other inflammatory cells such as lymphocytes and neutrophils,all of which can cause damage to islet cells. Thus, it is desirable tobegin therapy at the stage of macrophage activation.

TABLE I Quantitative Immunohistochemistry of PDE2 and PDE5 in Islet CellRegion Mean Number of Mean Number of Cells with Brown Cells with BrownIntensity in Islet Intensity in Islet Tissue Cells Cells IdentificationStage of Disease (PDE2) (PDE5) B14A Chronic Type-I 85 ± 32.9 111 ± 14.5B14D B14A age-matched 71 ± 32.6 87 ± 7.2 resistant control B14B AcuteType-I 56 ± 26.5 96 ± 6.8 B14E B14B age-matched 78 ± 6.3  102 ± 8.5 resistant control B14C Prone Type-I 58 ± 33.2 87 ± 6.4 B14F B14Cage-matched 57 ± 39.8 93 ± 4.9 resistant control

TABLE II Quantitative Immunohistochemistry of Positive StainedMacrophage in Islet Cell Region Total Number of Macrophages Tissue PerSix Identification Stage of Disease Pancreatic Islets B14A ChronicType-I 7 B14D B14A age-matched resistant control 0 B14B Acute Type-I 39*B14E B14B age-matched resistant control 0 B14C Prone Type-I 2 B14F B14Cage-matched resistant control 0 *compared to other groups, p value lessthan 0.001

Human Pancreatic Tissue PDE Analysis in Type I Diabetes Patients

Human formalin-fixed paraffin-embedded 5-μm thick pancreatic tissue wasobtained from a 22 year-old female and another female of unknown age.Both patients had a history of Type-I diabetes mellitus. A serialdilution study demonstrated the optimal signal-to-noise ratio was 1:100and 1:200 (PDE2), 1:500 and 1:1000 (PDE5). Anti-PDE2 and anti-PDE5antibodies were used as the primary antibodies, and the principaldetection system consisted of a Vector anti-sheep secondary (BA-6000)and Vector ABC-AP Kit (AK-5000) with a Vector Red substrate kit(SK-5100), which was used to produce a fuchsia-colored red deposit.Tissues were also stained with a positive control antibody (CD31) toensure the tissue antigens were preserved and accessible forimmunohistochemical analysis. CD31 is present in monocytes, macrophages,granulocytes, B lymphocytes and platelets. The negative controlconsisted of performing the entire immunohistochemistry procedure onadjacent sections in the absence of primary antibody. Slides were imagedusing a DVC Digital Photo Camera coupled to a Nikon microscope.

Human pancreatic tissue samples (1 and 2) exhibited positive stainingfor PDE2 and PDE5 proteins and immunostaining was mostly localized topancreatic islet cells. In FIGS. 17a and 17 b, pancreatic tissuespecimens from the two human patients described above are stained forPDE2 protein. In FIGS. 18a and 18 b pancreatic tissue specimens from thetwo human patients described above are stained for PDE5 protein.

FIG. 17b shows a positively stained macrophage overexpressing PDE5protein (see arrow).

It will be understood that various changes and modifications can be madein the details of procedure, formulation and use without departing fromthe spirit of the invention, especially as defined in the followingclaims.

We claim:
 1. A method of treating type I diabetes in a mammal with thatdisease comprising administering to the mammal a physiologicallyeffective amount of an inhibitor of PDE2.
 2. The method of claim 1wherein mammal is also administered an inhibitor of PDE5.
 3. The methodof claim 2 wherein said inhibitor of PDE2 and PDE5 comprise the samecompound.
 4. The method of claim 1 wherein said inhibitor does notsubstantially inhibit COX I or COX II.
 5. The method of claim 2 whereinsaid inhibitor does not substantially inhibit COX I or COX II.
 6. Themethod of claim 5 wherein said inhibitor has an IC₅₀ for PDE2 of no morethan about 25 μM. and has an IC₅₀ for each of the COX enzymes greaterthan about 40 μM.
 7. A method of treating type I diabetes in a mammalcomprising administering to the mammal a compound of the formula:

wherein R¹ is independently selected in each instance from the groupconsisting of hydrogen, halogen, lower alkyl, loweralkoxy, amino,loweralkylamino, di-loweralkylamino, loweralkylmercapto, loweralkylsulfonyl, cyano, carboxamide, carboxylic acid, mercapto, sulfonic acid,xanthate and hydroxy; R₂ is selected from the group consisting ofhydrogen and lower alkyl; R₃ is selected from the group consisting ofhydrogen, halogen, amino, hydroxy, lower alkyl amino, anddi-loweralkylamino; R₄ is hydrogen, or R₃ and R₄ together are oxygen; R₅and R₆ are independently selected from the group consisting of hydrogen,lower alkyl, hydroxy-substituted lower alkyl, amino lower alkyl, loweralkylamino-lower alkyl, lower alkyl amino di-lower alkyl, lower alkylnitrile, —CO₂H, —C(O)NH₂, and a C₂ to C₆ amino acid; R₇ is independentlyselected in each instance from the group consisting of hydrogen, aminolower alkyl, lower alkoxy, lower alkyl, hydroxy, amino, lower alkylamino, di-lower alkyl amino, amino lower alkyl, halogen, —CO₂H, —SO₃H,—SO₂NH₂, and —SO₂(lower alkyl); m and n are integers from 0 to 3independently selected from one another; Y is selected from the groupconsisting of quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl,pyrazinyl, imidazolyl, indolyl, benzimidazolyl, triazinyl, tetrazolyl,thiophenyl, furanyl, thiazolyl, pyrazolyl, or pyrrolyl, or substitutedvariants thereof wherein the substituents are one or two selected fromthe group consisting of halogen, lower alkyl, lower alkoxy, amino, loweralkylamino, di-lower alkylamino, hydroxy, —SO₂ (lower alkyl) and—SO₂NH₂; and pharmaceutically acceptable salts thereof.
 8. The method ofclaim 7 wherein Y is selected from pyridinyl or quinolonyl.
 9. Themethod of claim 7 wherein R₁ is selected from the group consisting ofhalogen, lower alkoxy, amino, hydroxy, lower alkylamino anddi-loweralkylamino.
 10. The method of claim 9 wherein R¹ is selectedfrom the group consisting of halogen, lower alkoxy, amino and hydroxy.11. The method of claim 7 wherein R₂ is lower alkyl.
 12. The method ofclaim 10 wherein R₂ is lower alkyl.
 13. The method of claim 7 wherein R₃is selected from the group consisting of hydrogen, halogen, hydroxy,amino, lower alkylamino and di-loweralkylamino.
 14. The method of claim10 wherein R₃ is selected from the group consisting of hydrogen,halogen, hydroxy, amino, lower alkylamino and di-loweralkylamino. 15.The method of claim 14 wherein R₃ is selected from the group consistingof hydrogen, hydroxy and lower alkylamino.
 16. The method of claim 14wherein R₃ is selected from the group consisting of hydrogen, hydroxyand lower alkylamino.
 17. The method of claim 7 wherein R₅ and R₆ areindependently selected from the group consisting of hydrogen,hydroxy-substituted lower alkyl, amino lower alkyl, loweralkylamino-lower alkyl, lower alkyl amino di-lower alkyl, —CO₂H,—C(O)NH₂.
 18. The method of claim 16 wherein R₅ and R₆ are independentlyselected from the group consisting of hydrogen, hydroxy-substitutedlower alkyl, amino lower alkyl, lower alkylamino-lower alkyl, loweralkyl amino di-lower alkyl, —CO₂H, —C(O)NH₂.
 19. The method of claim 7wherein R₅ and R₆ are independently selected from the group consistingof hydrogen, hydroxy-substituted lower alkyl, lower alkyl amino di-loweralkyl, —CO₂H, —C(O)NH₂.
 20. The method of claim 18 wherein R₅ and R₆ areindependently selected from the group consisting of hydrogen,hydroxy-substituted lower alkyl, lower alkyl amino di-lower alkyl,—CO₂H, —C(O)NH₂.
 21. The method of claim 7 wherein R₇ is independentlyselected in each instance from the group consisting of hydrogen, loweralkoxy, hydroxy, amino, lower alkyl amino, di-lower alkyl amino,halogen, —CO₂H, —SO₃H, —SO₂NH₂, amino lower alkyl, and —SO₂(loweralkyl).
 22. The method of claim 20 wherein R₇ is independently selectedin each instance from the group consisting of hydrogen, lower alkoxy,hydroxy, amino, lower alkyl amino, di-lower alkyl amino, halogen, —CO₂H,—SO₃H, —SO₂NH₂, amino lower alkyl, and —SO₂(lower alkyl).
 23. The methodof claim 7 wherein R₇ is independently selected in each instance fromthe group consisting of hydrogen, lower alkoxy, hydroxy, amino, halogen,—CO₂H, —SO₃H, —SO₂NH₂, amino lower alkyl, and —SO₂(lower alkyl).
 24. Themethod of claim 19 wherein R₇ is independently selected in each instancefrom the group consisting of hydrogen, lower alkoxy, hydroxy, amino,halogen, —CO₂H, —SO₃H, —SO₂NH₂, amino lower alkyl, and —SO₂(loweralkyl).
 25. The method of claim 23 wherein at least one of the R₇substituents is ortho- or para-located.
 26. She method of claim 24wherein at least one of the R₇ substituents is ortho- or para-located.27. The method of claim 25 wherein at least one of the R₇ substituentsis ortho-located.
 28. The method of claim 26 wherein at least one of theR₇ substituents is ortho-located.
 29. The method of claim 7 wherein Y isselected from the group consisting of quinolinyl, isoquinolinyl,pyridinyl, pyrimidinyl and pyrazinyl or said substituted variantsthereof.
 30. The method of claim 7 wherein said compound comprises(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride.
 31. The method of claim 7 wherein said compound comprises(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamidep-methylbenzenesulfonate.
 32. A method of inhibiting activatedmacrophages in a mammal with type I diabetes comprising chronicallyadministering to the mammal a physiologically effective amount of aninhibitor of PDE2.
 33. The method of claim 32 wherein mammal is alsoadministered an inhibitor of PDE5.
 34. The method of claim 33 whereinsaid inhibitor of PDE2 and PDE5 comprise the same compound.
 35. Themethod of claim 32 wherein said inhibitor does not substantially inhibitCOX I or COX II.
 36. The method of claim 34 wherein said inhibitor doesnot substantially inhibit COX I or COX II.
 37. The method of claim 32wherein the mammal is a companion pet.
 38. The method of claim 37wherein the mammal is human.
 39. A method of treating type I diabetes ina mammal with that disease comprising inhibiting PDE2 in the diseasedtissue without substantially inhibiting COX I or COX II.
 40. A method ofinhibiting activated macrophages in a mammal with type I diabetescomprising chronically administering to the mammal a physiologallyeffective amount of an inhibitor of PDE2 having a PDE2 IC₅₀ no more thanabout 25 μM and having a COX IC₅₀ greater than about 40 μM.